Delineation of the population genetic structure of Culicoides imicola in East and South Africa

BACKGROUND: Culicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region. METHODS: A panel of 12 DNA microsatellite markers isolated de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges. RESULTS: The microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth. CONCLUSIONS: The gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-1277-4) contains supplementary material, which is available to authorized users

Diagnosis and molecular epidemiology of the African horsesickness virus by the polymerase chain reaction and restriction patterns

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Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments.

&lt;p&gt;The detection of the bluetongue virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C(t) values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C(t) values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV.&lt;/p&gt;</p

Incursion of Bluetongue in Tunisia: Molecular Characterization of Viral Strains

In December 1999, the bluetongue was declared in Tunisia. Other cases of the disease have also been reported in other countries in the Mediterranean basin. The aim of this work was to characterize viral isolates obtained during the epizootic, which occurred from December 1999 to March 2000, and to perform serotype determination. The three genomic segments, 2, 7 and 10, corresponding to the proteins VP2, VP7 and NS3/NS3A, were amplified by PCR and sequenced. Sequences of the various genomic segments obtained from Tunisian viral isolates were compared with the Corsican vaccinal and wild-type strain, as well as with other bluetongue virus strains published in GenBank. The results are presented in the form of phylogenetic trees and tables in which nucleotide sequences are compared, thus showing the common origin of the Tunisian viral strain (serotype 2) and the Corsican strain that was responsible for the October 2000 epizootic

Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR.

&lt;p&gt;Real-time RT-PCR (RT-qPCR) was used routinely for laboratory diagnosis during the 2006/2007 bluetongue virus (BTV) serotype 8 epidemic. In the present study the impact of pooling and multiplexing strategies on RT-qPCR are assessed. To avoid any bias in the pooling experiments, 121 BTV-8 positive blood samples with a low to high viral load were selected and pooled individually with nine negative blood samples. Analyses of the individually and pooled samples indicated an overall mean difference of 4.32 Ct-values. The most pronounced differences were observed in samples with the lowest viral load of which 70% could no longer be detected after pooling. The pooling strategy is therefore not suitable for BTV detection at the individual level since animals infected recently may be missed. An alternative approach to reduce costs and workload is to apply a multiplexing strategy in which the viral RNA and internal beta-actin control RNA are detected in a single reaction. Parallel analysis (singleplex versus multiplex) of a 10-fold dilution series and 546 field samples proved that the sensitivity of the BTV RT-qPCR was not affected whereas the beta-actin reaction was reduced only slightly. Without the use of an internal control, 0.6% of 1985 field samples is at risk of being diagnosed incorrectly as negative.&lt;/p&gt;</p