Comparison of 2016–17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016–17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016–17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005–06 and 2016–17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016–17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control

Novel Quantitative Real-Time LCR for the Sensitive Detection of SNP Frequencies in Pooled DNA: Method Development, Evaluation and Application

BACKGROUND: Single nucleotide polymorphisms (SNP) have proven to be powerful genetic markers for genetic applications in medicine, life science and agriculture. A variety of methods exist for SNP detection but few can quantify SNP frequencies when the mutated DNA molecules correspond to a small fraction of the wild-type DNA. Furthermore, there is no generally accepted gold standard for SNP quantification, and, in general, currently applied methods give inconsistent results in selected cohorts. In the present study we sought to develop a novel method for accurate detection and quantification of SNP in DNA pooled samples. METHODS: The development and evaluation of a novel Ligase Chain Reaction (LCR) protocol that uses a DNA-specific fluorescent dye to allow quantitative real-time analysis is described. Different reaction components and thermocycling parameters affecting the efficiency and specificity of LCR were examined. Several protocols, including gap-LCR modifications, were evaluated using plasmid standard and genomic DNA pools. A protocol of choice was identified and applied for the quantification of a polymorphism at codon 136 of the ovine PRNP gene that is associated with susceptibility to a transmissible spongiform encephalopathy in sheep. CONCLUSIONS: The real-time LCR protocol developed in the present study showed high sensitivity, accuracy, reproducibility and a wide dynamic range of SNP quantification in different DNA pools. The limits of detection and quantification of SNP frequencies were 0.085% and 0.35%, respectively. SIGNIFICANCE: The proposed real-time LCR protocol is applicable when sensitive detection and accurate quantification of low copy number mutations in DNA pools is needed. Examples include oncogenes and tumour suppressor genes, infectious diseases, pathogenic bacteria, fungal species, viral mutants, drug resistance resulting from point mutations, and genetically modified organisms in food

Comparison of 2016–17 and Previous Epizootics of Highly Pathogenic Avian Influenza H5 Guangdong Lineage in Europe

We analyzed the highly pathogenic avian influenza (HPAI) H5 epizootic of 2016–17 in Europe by epidemiologic and genetic characteristics and compared it with 2 previous epizootics caused by the same H5 Guangdong lineage. The 2016–17 epizootic was the largest in Europe by number of countries and farms affected and greatest diversity of wild birds infected. We observed significant differences among the 3 epizootics regarding region affected, epidemic curve, seasonality, and outbreak duration, making it difficult to predict future HPAI epizootics. However, we know that in 2005–06 and 2016–17 the initial peak of wild bird detections preceded the peak of poultry outbreaks within Europe. Phylogenetic analysis of 2016–17 viruses indicates 2 main pathways into Europe. Our findings highlight the need for global surveillance of viral changes to inform disease preparedness, detection, and control

Incidence of viruses infecting spinach in Greece, highlighting the importance of weeds as reservoir hosts

The aim of this survey was to identify viruses infecting spinach (Spinacia oleracea L.) in the most important spinach-producing areas in Greece. A total of 1074 spinach samples were collected from eleven districts belonging to the prefectures of Thessaloniki, Chalkidiki and Imathia in northern Greece, and Evia in central Greece. Samples were tested by ELISA, mechanical inoculation onto indicator plants and immunoelectron microscopy. Beet western yellows virus (BWYV), Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) were identified in 13.5%, 7% and 5.4% of samples, respectively, infected samples being detected in all regions examined. This is the first record of CMV and TuMV infecting spinach in Greece, and the first report of BWYV occurrence in any crop nationwide. Surveys were also conducted to assess the potential reservoir hosts of BWYV, CMV and TuMV in weeds collected from spinach fields. All three viruses were detected among 125 samples tested by ELISA. TuMV prevailed as it occurred in 14.4% of all weed sample

First report of Tomato chlorosis virus on tomato crops in Cyprus

In the summer of 2004, yellowing symptoms similar to those caused by nitrogen and/or magnesium deficiency were observed in field- and glasshouse-grown tomatoes ( Lycopersicon esculentum ), in the Parekklisia area of Cyprus. Initially, lower leaves showed extensive interveinal yellowing with necrotic flecks, brittleness and occasional upward leaf rolling, before finally the whole plant turned yellow. Similar symptoms were observed during 2005 in glasshouse tomatoes grown in areas located on the southwest coastal region of the island. The abundance of whiteflies on the affected plants suggested the involvement of the whitefly-transmitted Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV), both of the genus Crinivirus (Wisler et al ., 1998). Leaves of 18 affected plants were collected, total RNA was isolated and RT-PCR was performed in a single tube using primers HS-11 and HS-12, followed by a multiplex nested-PCR with primers TIC-3/TIC-4 and ToC- 5/ToC-6, for the detection of TICV and ToCV, respectively (Dovas et al ., 2002). A PCR product of 463 bp, corresponding to the HSP 70 gene of ToCV, was amplified for all tested samples. The sequences of four cloned PCR products were identical (EMBL accession number AM158958) and showed 99% nucleotide identity to a ToCV isolate from Florida (accession number AY903448). ToCV is vectored by Bemisia tabaci ( biotypes A and B ), Trialeurodes vaporariorum and T. abutilonea . Although there have been no systematic studies on whitefly incidence and distribution in Cyprus, it seems that B. tabaci is the predominant species present, as Tomato yellow leaf curl virus (Ioannou, 1985) and Cucurbit yellow stunting disorder virus (Papayiannis et al ., 2005), vectored by this species, are prevalent in tomatoes and cucurbit crops, respectively. On the other hand, the incidence of Beet pseudo-yellows virus (transmitted by T. vaporariorum) is much lower. This is the first report of ToCV in Cyprus

First report of Tomato chlorosis virus on tomato crops in Cyprus

In the summer of 2004, yellowing symptoms similar to those caused by nitrogen and/or magnesium deficiency were observed in field- and glasshouse-grown tomatoes ( Lycopersicon esculentum ), in the Parekklisia area of Cyprus. Initially, lower leaves showed extensive interveinal yellowing with necrotic flecks, brittleness and occasional upward leaf rolling, before finally the whole plant turned yellow. Similar symptoms were observed during 2005 in glasshouse tomatoes grown in areas located on the southwest coastal region of the island. The abundance of whiteflies on the affected plants suggested the involvement of the whitefly-transmitted Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV), both of the genus Crinivirus (Wisler et al ., 1998). Leaves of 18 affected plants were collected, total RNA was isolated and RT-PCR was performed in a single tube using primers HS-11 and HS-12, followed by a multiplex nested-PCR with primers TIC-3/TIC-4 and ToC- 5/ToC-6, for the detection of TICV and ToCV, respectively (Dovas et al ., 2002). A PCR product of 463 bp, corresponding to the HSP 70 gene of ToCV, was amplified for all tested samples. The sequences of four cloned PCR products were identical (EMBL accession number AM158958) and showed 99% nucleotide identity to a ToCV isolate from Florida (accession number AY903448). ToCV is vectored by Bemisia tabaci ( biotypes A and B ), Trialeurodes vaporariorum and T. abutilonea . Although there have been no systematic studies on whitefly incidence and distribution in Cyprus, it seems that B. tabaci is the predominant species present, as Tomato yellow leaf curl virus (Ioannou, 1985) and Cucurbit yellow stunting disorder virus (Papayiannis et al ., 2005), vectored by this species, are prevalent in tomatoes and cucurbit crops, respectively. On the other hand, the incidence of Beet pseudo-yellows virus (transmitted by T. vaporariorum) is much lower. This is the first report of ToCV in Cyprus

Incidence of Viruses Infecting Cucurbits in Cyprus

In a survey during 2000-2002 to determine the identity and prevalence of viruses affecting cucurbit crops in Cyprus, 2993 samples of cucumber, zucchini, melon and watermelon were collected from the five major cucurbit-growing areas in Cyprus. Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus type W (PRSV-W), Watermelon mosaic virus (WMV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV) and Squash mosaic virus (SqMV) were detected by enzyme-linked immunosorbent assay (ELISA), and Cucurbit yellow stunting disorder virus (CYSDV), Beet pseudo-yellows virus (BPYV) and Cucumber vein yellowing virus (CVYV) by reverse transcription polymerase chain reaction (RT-PCR). ZYMV was the most prevalent virus of cucurbits in Cyprus with an overall incidence of 45%. PRSV-W, CABYV and WMV were detected in 20.8%, 20.8% and 7.8% of the samples tested, respectively. CYSDV was detected in most greenhouse cucumber samples with yellowing symptoms (88.1%), whereas BPYV and CVYV were found in only 2.4% and 9.5%, respectively, of samples. CMV and SqMV were not detected in any cucurbitaceous crop during this survey

Isolation of <it>Mycobacterium avium subspecies paratuberculosis</it> from Ugandan cattle and strain differentiation using optimised DNA typing techniques

Abstract Background The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda. Results Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2). Conclusions There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.</p