Reverse Hybridization Assay Research Articles

Clinical Characteristics and Genetic Variations in Early-Onset Atopic Dermatitis Patients.

BackgroundHereditary factors contribute to atopic dermatitis (AD) development. We developed the reverse blot hybridization assay (REBA) kit to simultaneously detect variations in skin barrier- and immune response-related genes prevalent in Korean AD patients.ObjectiveTo identify genetic variations and clinical characteristics that could predict early AD development.MethodsWe compared AD-related genetic variations between early-onset AD subjects and non-AD controls, and clinical characteristics and genetic variations between early- and late-onset AD subjects. We compared 28 early-onset AD subjects and 57 non-AD controls from a birth cohort and 108 early- (age ≤3 years) and 90 late-onset AD subjects and 189 non-AD controls from a university hospital. Genetic variations were detected via REBA.ResultsThere were no differences in AD-related genetic variation between early-onset AD subjects and non-AD controls in the birth cohort. When the birth cohort and hospital populations were combined, early-onset AD subjects and non-AD controls showed different frequencies of genetic variations of KLK7, SPINK5 1156, DEFB1, IL5RA, IL12RB1a, and IL12RB1b. No differences in the frequency of genetic variations were observed between early- and late-onset AD subjects. Immunoglobulin E positivity for house dust mites was prevalent in late-onset AD subjects. A family history of atopic diseases was associated with early-onset AD.ConclusionNo AD-related genetic variations could predict early AD development in Koreans, even though neonates with a family history of atopic diseases are likely to develop AD at ≤3 years of age. Environmental exposure may be more important than genetic variation in determining the onset age of AD.

Human papillomavirus (HPV) prevalence and associated risk factors in women from Curaçao.

BackgroundIn the Caribbean region, a notable difference in HPV-prevalence and genotypes distribution between the islands is observed. Recently we found in Curaçao a low incidence of HPV16 and 18 in cervical cancer compared to the standard world population. We aimed to determine HPV-prevalence, HPV-genotype distribution and associated risk-factors in women from Curaçao.Methods5000 women aged 25–65 years were randomly selected from the national Population Register. HPV was detected by means of GP5+/6+PCR EIA and GP 5+/6+amplimers from HPV-positive samples were genotyped with a reverse hybridisation assay. We also collected personal data and data on risk-factors.Results1075 women were enrolled in the study. Overall HPV-prevalence was 19.7%. Most frequent genotypes were HPV16 (2.3%), 35 (2.1%) and 52 (1.8%). Twenty-seven women detected with abnormal cytology (i.e.≥ASC-US) were referred for biopsy. In women with normal cytology (n = 1048), HPV-prevalence was 17.9% and the most common high-risk HPV (hrHPV)-types were HPV35 (2.0%), 18 (1.8%), 16 (1.5%) and 52 (1.5%). The highest HPV-prevalence (32.8%) was found in the age-group: 25–34 (n = 247). HPV positive women started sex at a younger age (p = 0.032).ConclusionsHPV-prevalence in the overall population is high and HPV16 was the most common genotype followed by 35 and 18. In women with normal cytology HPV35 is the most common genotype followed by HPV18, 52 and 16. The high HPV-prevalence (32.8%) in women of 25–34 years argue for introduction of cervical cancer prevention strategies. HPV-type distribution found in Curaçao should be taken into account when considering the choice for prophylactic vaccination.

EGFR Mutational Profiling in Non-Small Cell Lung Cancer: The Clinical Performance of a Sensitive Reverse-Hybridization Assay.

In patients with non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutations have been associated with the tumor response to targeted therapy with EGFR tyrosine kinase inhibitors. Although labor intensive and not very sensitive (ie, an analytical sensitivity of 20%), direct sequencing is widely used for mutation detection. This study aimed at evaluating the potential of a test strip-based reverse-hybridization assay (EGFR StripAssay), designed for the simultaneous detection of 16 mutations in exons 18 to 21 of the EGFR gene, to sensitively identify EGFR mutation in DNA from NSCLC tissue samples. Formalin-fixed paraffin-embedded (FFPE) DNA samples from 59 patients with a histologically confirmed primary NSCLC tumor were used to compare the performance of the EGFR StripAssay against that of the Sanger sequencing. The EGFR StripAssay analysis identified 7 (11.8%) of 59 FFPE samples to carry an EGFR mutation, of which 4 (57.1%) and 3 (42.8%) samples were positive for exon 19 and 21 mutations, respectively. Of note, no sample was identified with EGFR exon 18 or 20 mutation. All mutations were confirmed by DNA sequencing. Using 50 ng of template DNA, the EGFR StripAssay demonstrated a detection limit of 1% mutant sequence in a background of normal DNA. The EGFR StripAssay is a fast and robust platform for the sensitive detection of EGFR mutation in FFPE DNA. Therefore, this assay could be considered as an alternative protocol to Sanger sequencing for EGFR mutation testing on limited-quantity samples.

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.

Simultaneous detection of barrier- and immune-related gene variations in patients with atopic dermatitis by reverse blot hybridization assay.

Hereditary factors are involved in the pathogenesis of atopic dermatitis (AD). However, AD-related gene variations are significantly different across ethnicities. To identify mutations and single-nucleotide polymorphisms (SNPs) in barrier- or immune-related genes from Korean patients with AD and compare the variations with those observed in nonatopic healthy controls (HCs), and to use novel reverse blot hybridization assay (REBA) for AD-related gene variants. We carried out REBA to simultaneously detect variations in genes related to barrier or immune function, namely, FLG, SPINK5, KLK7, DEFB1, TNFα, KDR, FCER1A, IL4, IL5,IL5RA, IL9, IL10, IL12, IL12R, IL13 and IL18, from Korean patients with AD, and compared the variation to that in nonatopic healthy controls. The homozygous mutants of KLK7 and SPINK5-2475, and the heterozygous mutants of FLG 3321delA, SPINK5-1156, DEFB1, KDR, IL5RA, IL9 and IL12RB1 were significantly more frequent in AD. It has been predicted that the larger the number of gene variants, the higher the odds ratio of AD prevalence; however, we did not find any significant correlation between the number of gene variants and AD severity. Using REBA, we identified more genetic variants that can predict AD occurrence. We also verified that REBA can be used to easily and accurately detect multiple AD-related gene variants simultaneously. In addition, we identified a correlation between KLK7 mutation and AD in Koreans, which is the first such report, to our knowledge.

Analysis of Common β-Thalassemia Mutations in North Vietnam

Available and flexible choice of methods for screening and detecting β-thalassemia (β-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common β-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (–TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

The role of human papillomavirus in p16-positive oral cancers.

The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope® (a chromogenic RNA insitu hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC.

Comparison of a prototype reverse hybridization assay and MethyLight for detection of SFRP2 promotor methylation in fecal DNA.

This study aimed to evaluate the diagnostic performance of a novel nonquantitative methylation-specific reverse hybridization (MSRH) assay to detect secreted frizzled-related protein 2 (SFRP2) promotor methylation in fecal DNA. SFRP2 promoter methylation was investigated in stool DNA isolated from 18 colorectal cancer (CRC) patients and 22 healthy controls using the MSRH assay based on methylation-specific DNA amplification followed by reverse hybridization of biotinylated amplicons to sequence-specific methylation detection probes, with MethyLight serving as a reference method. SFRP2 promotor methylation as determined by MSRH vs. MethyLight showed a sensitivity and specificity of 61.1% and 86.3% vs. 77.7% and 77.3%, respectively. Moderate agreement (ĸ = 0.54, 95% confidence interval [95% CI], 0.29-0.80, p<0.001) was observed between the 2 methods. However, the differences in SFRP2 promotor methylation observed between CRC patients and healthy individuals by both assays were statistically significant (p<0.001). Our findings, although limited by the small sample size, do not support the use of the MSRH assay for CRC screening in stool.

The spectrum of Familial Mediterranean Fever gene (MEFV) mutations and genotypes in Iran, and report of a novel missense variant (R204H)

BackgroundFamilial Mediterranean Fever (FMF) is an autosomal recessive disorder, characterized by recurrent and self-limited episodes of fever, abdominal pain, synovitis and pleuritis. FMF as the most common inherited monogenic autoinflammatory disease mainly affects ethnic groups of the Mediterranean basin, Arab, Jewish, Turkish, Armenian North Africans and Arabic descent. Materials and methodsIn the present study, we selected 390 unrelated FMF patients according to the Tel-Hashomer criteria, and analyzed all patients for 12 most common mutations of MEFV gene by reverse hybridization assay (FMF strip assay). We also investigated exon 2 and 10 of MEFV gene in 78 patients by Sanger sequencing. ResultsAccording to strip assay results, at least one mutation was found in 234 patients (60%), and no mutation was found in other 156 patients (40%). The five most common mutations and allelic frequencies were M694V (13.6%), E148Q (10.4%), M694I (6.5%), V726A (4.1%), and M680I (3.8%). Moreover, we detected a novel missense variant (R204H, c.611 G > A) (SCV000297822) and following rare mutations among sequenced samples; R202Q, P115T, G304R, and E230K. ConclusionThis study describes the MEFV mutations spectrum and distribution in Iranian population, and shows different mutation patterns among Iranian ethnicities. Moreover, M694V is the most common MEFV mutation in Iran.

P180 Moving onto plan C: Finding detours when DSA creates a roadblock to bone marrow transplantation

Aim This case study illustrates potential avenues to consider when identifying hematopoietic stem cell donor options for HLA sensitized patients. Methods HLA typing was performed using reverse sequence specific oligonucleotide probe hybridization assays (One Lambda, Inc.) and Sanger sequence base typing assays (local and commercial kits). HLA-specific antibody was assessed using class I and class II phenotypes (Lifecodes®, Immucor) and single antigen panels (One Lambda Inc.). Results The presence of donor specific antibody (DSA) can adversely impact the likelihood of bone marrow engraftment depending on the specificity and strength of the DSA. DSA assessment and monitoring is becoming crucial as centers are utilizing HLA mismatched donors. In the last 5 years, 60–70% of the overall allogeneic bone marrow transplants at our center utilized HLA mismatched donors. In a majority of our sensitized patients, the availability of multiple eligible donors allowed for either the avoidance of DSA or the minimization of DSA to levels that did not warrant desensitization. But, in 6% of the cases evaluated in 2016, DSA caused roadblocks requiring alternate pathways. The case of a 41 year old female with Acute Lymphocytic Leukemia illustrates the “Moving onto Plan C” mantra. Plan A: A related haploidentical transplant was diverted due to crossmatch positive DSA against the patient’s four eligible donors. Plan B: An unrelated matched transplant was deterred due to DRB3*01 allele specific DSA against several 10/10 HLA loci matched donors with the DRB1*13:01/DRB3*01 association instead of patient’s DRB1*13:01/DRB3*02 association. Plan C: An unrelated mismatched donor search was initiated to identify 9/10 HLA loci matched donors with a DRB1 mismatch likely to avoid the patient’s broad specificity and eliminate the potential of a DRB3*01 typed donor. Conclusion Even though there is a low incidence of patients requiring an alternate route due to DSA, the impact of the DSA is life-threatening to the individuals in this cohort. With the increase of HLA mismatched transplants, there is a greater need for initial HLA antibody assessments, continual HLA antibody monitoring, alternative back-up plans and creative solutions in order to offer all patients the best chance of survival.

AB130. Comprehensive analysis of CYP2D6 and copy number variants using reverse-hybridization and real-time PCR-based assays

AB130. Comprehensive analysis of CYP2D6 and copy number variants using reverse-hybridization and real-time PCR-based assays

Impact of updating the MALDI-TOF MS database on the identification of nontuberculous mycobacteria.

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI-TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow-growing mycobacteria and 108 of rapid-growing mycobacteria. MALDI-TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p=0.0863) isolates and 15 (36.6%; p=0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p<0.01). The scores obtained with MALDI-TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p<0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI-TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI-TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.

A Historical Overview of Research on Babesia orientalis, a Protozoan Parasite Infecting Water Buffalo.

Babesiosis is a globally important zoonotic disease caused by tick-borne intraerythrocytic protozoan of the genus Babesia (phylum apicomplexa). In China, there are five species that infect cattle buffalo and cause great economic loss, which include Babesia bigemina, B. bovis, B. major, B. ovata, and B. orientalis. Among them, B. orientalis is the most recently identified new Babesia species epidemic in China. This review summarized the work done in the past 33 years to give an overview of what learned about this parasite. This parasitic protozoan was found in 1984 in Central and South China and then named as B. orientalis in 1997 based on its differences in transmitting host, morphology, pathogenicity and characteristics of in vitro cultivation when compared with B. bigemina and B. bovis. It was found that Rhipicephalus haemaphysaloides is the transmitting vector and water buffalo is the only reported host. Phylogenetic analysis based on the 18S rRNA gene also confirmed that B. orientalis is a new species. After species verification, four diagnostic methods including semi-nest PCR, loop-mediated isothermal amplification assay, reverse line blot hybridization assay, and real-time PCR were established for lab and field use purposes. Genomic sequencing was conducted and the complete genomes of mitochondria and apicoplast were annotated. Future work will be focused on developing effective vaccines, identifying drug targets and screening useful drugs for controlling B. orientalis in water buffalo.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria

BackgroundThe differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. MethodsA total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. ResultsThe sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15–100%, p<0.0001) and 97.8% (95% CI 91.86–99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56–100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529–534, 524–529, and 514–520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). ConclusionsThe entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48–72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples.

Co-inheritance of glucose-6-phosphate dehydrogenase deficiency mutations and hemoglobin E in a Kachin population in a malaria-endemic region of Southeast Asia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobin E (HbE, β26 Glu-Lys) are two common red cell disorders in Southeast Asia. G6PD deficiency produces hemolytic anemia, which can be triggered by certain drugs or infections. HbE is asymptomatic or is manifested as microcytic, minimally hemolytic anemia. The association between G6PD deficiency and HbE is little understood. This study aimed to investigate G6PD deficiency and HbE in a Kachin ethnic group in the China-Myanmar border area. G6PD enzyme activity was measured using a quantitative G6PD assay, G6PD variants genotyped by the SNaPshot assay, and an HbE gene mutation identified by an amplification refractory mutation system and subsequently confirmed by using a reverse dot blot hybridization assay from 100 unrelated individuals in the study area. G6PD enzyme activity ranged from 0.4 to 24.7 U/g Hb, and six males had severe G6PD deficiency (<0.12–1.2 U/g Hb), while six males and 12 females had mild G6PD deficiency (>1.2–4.5 U/g Hb). Among the 24 G6PD-deficient subjects, 22 (92%) had the Mahidol 487G>A mutation (12 male hemizygotes, one female homozygote, and nine female heterozygotes), while the G6PD genotypes in two female subjects were unknown. HbE was identified in 39 subjects (20 males and 19 females), including 15 HbEE (seven males and eight females) and 24 HbAE (13 males and 11 females). Twenty-three subjects co-inherited both G6PD deficiency and HbE (22 with HbAE and one with HbEE). Whereas mean Hb levels were not significantly different between the HbA and HbE groups, G6PD-deficient males had significantly lower Hb levels than G6PD-normal males (P < 0.05, t-test). However, it is noteworthy that two G6PD-deficient hemizygous males with HbAE were severely anemic with Hb levels below 50 g/L. This study revealed high prevalence of co-inheritance of G6PD deficiency with HbAE in the Kachin ethnicity, and a potential interaction of the G6PD Mahidol 487G>A and HbAE in males leading to severe anemia. The presence of 6% males with severe G6PD deficiency raised a major concern in the use of primaquine for radical cure of vivax malaria.

Development of a Rapid Reverse Blot Hybridization Assay for Detection of Clinically Relevant Antibiotic Resistance Genes in Blood Cultures Testing Positive for Gram-Negative Bacteria.

Rapid and accurate identification of the causative pathogens of bloodstream infections is crucial for the prompt initiation of appropriate antimicrobial therapy to decrease the related morbidity and mortality rates. The aim of this study was to evaluate the performance of a newly developed PCR-reverse blot hybridization assay (REBA) for the rapid detection of Gram-negative bacteria (GNB) and their extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase resistance genes directly from the blood culture bottles. The REBA-EAC (ESBL, AmpC β-lactamase, carbapenemase) assay was performed on 327 isolates that were confirmed to have an ESBL producer phenotype, 200 positive blood culture (PBCs) specimens, and 200 negative blood culture specimens. The concordance rate between the results of REBA-EAC assay and ESBL phenotypic test was 94.2%. The sensitivity, specificity, positive predictive value, and negative predictive value of the REBA-EAC assay for GNB identification in blood culture specimens were 100% (95% CI 0.938–1.000, P < 0.001), 100% (95% CI 0.986–1.000, P < 0.001), 100% (95% CI 0.938–1.000, P < 0.001), and 100% (95% CI 0.986–1.000, P < 0.001), respectively. All 17 EAC-producing GNB isolates from the 73 PBCs were detected by the REBA-EAC assay. The REBA-EAC assay allowed easy differentiation between EAC and non-EAC genes in all isolates. Moreover, the REBA-EAC assay was a rapid and reliable method for identifying GNB and their β-lactamase resistance genes in PBCs. Thus, this assay may provide essential information for accelerating therapeutic decisions to achieve earlier appropriate antibiotic treatment during the acute phase of bloodstream infection.

Molecular evidence of a new Babesia sp. in goats

In this study, a novel Babesia sp. infecting goats was detected and its phylogenetic relationship to related species was determined. A total of 200 blood samples collected from sheep (n=78) and goats (n=122) were examined in the study. The V4 hypervariable region of the 18S rRNA gene of the novel Babesia sp. was amplified by PCR and analysed using a reverse line blot hybridization assay adapted for small ruminants. Samples from seven goats hybridized to Theileria/Babesia catch-all and Babesia catch-all probes and did not hybridize to any species-specific probe tested, suggesting the presence of an unrecognized Babesia species or genotype. Sequencing results showed the isolate to clearly differ from ovine Babesia species and genotypes currently available in the GenBank database. The isolate showed 90.9%, 93.5%, and 93.4% identity to B. ovis, B. motasi, and B. crassa, respectively and 91–93% similarity to Babesia genotypes recently described in small ruminants. The highest homology (∼96–97%) observed was with Babesia odocoilei, Babesia sp. EU1, and Babesia divergens. The new isolate was provisionally designated Babesia sp. The study contributes to better insight into the distribution and phylogenetic diversity of piroplasms in small ruminants. The survey indicated a high prevalence of piroplasms in small ruminants (21.5%). Of those detected, T. ovis was the most prevalent (17%), followed by Babesia sp. (3.5%), and B. ovis (2%).

A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization

An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent.

SURVEILLANCE FOR VIRAL AND PARASITIC PATHOGENS IN A VULNERABLE AFRICAN LION (PANTHERA LEO) POPULATION IN THE NORTHERN TULI GAME RESERVE, BOTSWANA.

African lion ( Panthera leo ) numbers are decreasing rapidly and populations are becoming smaller and more fragmented. Infectious diseases are one of numerous issues threatening free-ranging lion populations, and low-density populations are particularly at risk. We collected data on the prevalence and diversity of viral and parasitic pathogens in a small lion population in eastern Botswana. During 2012 and 2014, blood samples were collected from 59% (n=13) of the adult-subadult lions in the Northern Tuli Game Reserve in eastern Botswana. One lion had antibodies to feline panleukopenia virus, two had antibodies to canine distemper virus, and two had feline calicivirus antibodies. Ten of the 13 had antibodies to feline immunodeficiency virus and 11 had feline herpesvirus antibodies. All lions were negative for antibodies to feline coronavirus. Blood samples from all lions were negative for Trypanosoma, Anaplasma, Theileria, and Ehrlichia spp. by molecular testing; however, all lions were positive for Babesia spp. by reverse line blot hybridization assay. Sequencing of amplicons from four lions revealed four groups of Babesia spp. including several genetic variants of Babesia felis , Babesia lengau, and Babesia canis and a group of novel Babesia sequences which were only 96% similar to other Babesia spp. Six lions were infested with four species of ticks (Rhipicentor nuttalli, Rhipicephalus simus, Rhipicephalus sulcatus, and Rhipicephalus appendiculatus). These data provide the first health assessment of this population and can be used to identify management and conservation strategies to decrease the impact of pathogens on this population. This is particularly important as there is an initiative to incorporate this population into a larger metapopulation of lions from adjacent South Africa and Zimbabwe.

261 Variations of genes related with skin barrier or immune response in the patients with atopic dermatitis could be simultaneously and rapidly detected on a membrane for reverse blot hybridization assay

261 Variations of genes related with skin barrier or immune response in the patients with atopic dermatitis could be simultaneously and rapidly detected on a membrane for reverse blot hybridization assay