CD8+ T-cells mediate immunopathology in tick-borne encephalitis

Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8(+) T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8(-/-) mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8(+) T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection. (C) 2008 Elsevier Inc. All rights reserved.

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

Streptococcus devriesei sp nov., from equine teeth

Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, cc-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a close affinity with Streptococcus mutans, Streptococcus ferus and related organisms. Sequence divergence values of > 5% with thew and other reference streptococcal species however demonstrated the organisms from equine sources represent a novel species. Based on the phenotypic distinctiveness of the new bacterium and molecular chemical and molecular genetic evidence, it is proposed that the unknown species be classified as Streptococcus devriesei sp. nov. The type strain of Streptococcus devriesei is CCUG 47155(T) (= CIP 107809T).

Equine anthelmintics: survey of the patterns of use, beliefs and attitudes among horse owners in the UK

An online survey was conducted to establish horse owners' beliefs, attitudes and practices relating to the use of anthelmintic drugs. Out of a total of 574 respondents, 89 per cent described themselves as ‘leisure riders’, most of whom took part in a variety of activities including eventing, show jumping, dressage, hunter trials, hunting, driving, endurance and showing. Overall, respondents were generally aware and concerned about the issue of anthelmintic resistance. Less than 60 per cent of all respondents were comfortable with their existing anthelmintic programme, and 25 per cent would like to reduce the use of anthelmintics in their horses. Of all the respondents, 47 per cent used livery, and 49 per cent of those reported that the livery imposed a common anthelmintic programme for horses kept on the premises; 45 per cent of these respondents were not entirely happy with the livery yard's programme. Less than 50 per cent of all respondents included ‘veterinary surgeon’ among their sources of advice on worming.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

The interannual variability of energy transports within and over the Atlantic Ocean in a coupled climate model

To gain a new perspective on the interaction of the Atlantic Ocean and the atmosphere, the relationship between the atmospheric and oceanic meridional energy transports is studied in a version of HadCM3, the U.K. Hadley Centre's coupled climate model. The correlation structure of the energy transports in the atmosphere and Atlantic Ocean as a function of latitude, and the cross correlation between the two systems are analyzed. The processes that give rise to the correlations are then elucidated using regression analyses. In northern midlatitudes, the interannual variability of the Atlantic Ocean energy transport is dominated by Ekman processes. Anticorrelated zonal winds in the subtropics and midlatitudes, particularly associated with the North Atlantic Oscillation (NAO), drive anticorrelated meridional Ekman transports. Variability in the atmospheric energy transport is associated with changes in the stationary waves, but is only weakly related to the NAO. Nevertheless, atmospheric driving of the oceanic Ekman transports is responsible for a bipolar pattern in the correlation between the atmosphere and Atlantic Ocean energy transports. In the Tropics, the interannual variability of the Atlantic Ocean energy transport is dominated by an adjustment of the tropical ocean to coastal upwelling induced along the Venezuelan coast by a strengthening of the easterly trade winds. Variability in the atmospheric energy transport is associated with a cross-equatorial meridional overturning circulation that is only weakly associated with variability in the trade winds along the Venezuelan coast. In consequence, there is only very limited correlation between the atmosphere and Atlantic Ocean energy transports in the Tropics of HadCM3

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.

Burkholderia Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

BACKGROUND: The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei. RESULTS: Using bacteriophage-mediated immunoscreening we identified genes expressed in vivo during experimental equine glanders infection. A family of immunodominant antigens were identified that share protein domain architectures with hemagglutinins and invasins. These have been designated Burkholderia Hep_Hag autotransporter (BuHA) proteins. A total of 110/207 positive clones (53%) of a B. mallei expression library screened with sera from two infected horses belonged to this family. This contrasted with 6/189 positive clones (3%) of a B. pseudomallei expression library screened with serum from 21 patients with culture-proven melioidosis. CONCLUSION: Members of the BuHA proteins are found in other Gram-negative bacteria and have been shown to have important roles related to virulence. Compared with other bacterial species, the genomes of both B. mallei and B. pseudomallei contain a relative abundance of this family of proteins. The domain structures of these proteins suggest that they function as multimeric surface proteins that modulate interactions of the cell with the host and environment. Their effect on the cellular immune response to B. mallei and their potential as diagnostics for glanders requires further study.

Corynebacterium uterequi sp. nov., a non-lipophilic bacterium isolated from urogenital samples from horses

Three strains of a Gram-positive, catalase-positive, fermentative, non-lipophilic, previously unknown bacterium were isolated from urogenital samples taken from mares in Scotland (M401624/00/1) and Sweden (VM 2074 and VM 2298T). All were deposited with the CCUG with tentative identifications as Corynebacterium spp. The strains were characterized using a polyphasic taxonomic approach. Biochemically, the strains were very similar to each other, but phylogenetically distinct from Corynebacterium species with validly published names (≤95% sequence similarity). rpoB gene sequence data confirmed the strains belonged to the same species (>99% sequence similarity) and were distinct from species with validly published names (>13% sequence divergence). On the basis of phenotypic and sequence data, the strains represent a novel species within the genus Corynebacterium, for which the name Corynebacterium uterequi is proposed. The type strain is VM 2298T (=CCUG 61235T = DSM 45634T), isolated from equine uterus.

Dominant components of the Thoroughbred metabolome characterised by 1H‐NMR spectroscopy: a metabolite atlas of common biofluids

Summary Reasons for performing study: Metabonomics is emerging as a powerful tool for disease screening and investigating mammalian metabolism. This study aims to create a metabolic framework by producing a preliminary reference guide for the normal equine metabolic milieu. Objectives: To metabolically profile plasma, urine and faecal water from healthy racehorses using high resolution 1H-NMR spectroscopy and to provide a list of dominant metabolites present in each biofluid for the benefit of future research in this area. Study design: This study was performed using seven Thoroughbreds in race training at a single time-point. Urine and faecal samples were collected non-invasively and plasma was obtained from samples taken for routine clinical chemistry purposes. Methods: Biofluids were analysed using 1H-NMR spectroscopy. Metabolite assignment was achieved via a range of 1D and 2D experiments. Results: A total of 102 metabolites were assigned across the three biological matrices. A core metabonome of 14 metabolites was ubiquitous across all biofluids. All biological matrices provided a unique window on different aspects of systematic metabolism. Urine was the most populated metabolite matrix with 65 identified metabolites, 39 of which were unique to this biological compartment. A number of these were related to gut microbial host co-metabolism. Faecal samples were the most metabolically variable between animals; acetate was responsible for the majority (28%) of this variation. Short chain fatty acids were the predominant features identified within this biofluid by 1H-NMR spectroscopy. Conclusions: Metabonomics provides a platform for investigating complex and dynamic interactions between the host and its consortium of gut microbes and has the potential to uncover markers for health and disease in a variety of biofluids. Inherent variation in faecal extracts along with the relative abundance of microbial-mammalian metabolites in urine and invasive nature of plasma sampling, infers that urine is the most appropriate biofluid for the purposes of metabonomic analysis.