Veterinary Biosciences - Research Publications

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    Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila
    Kurscheid, S ; Lew-Tabor, AE ; Valle, MR ; Bruyeres, AG ; Doogan, VJ ; Munderloh, UG ; Guerrero, FD ; Barrero, RA ; Bellgard, MI (BMC, 2009-03-26)
    BACKGROUND: The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. RESULTS: We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. CONCLUSION: We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.
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    Determining Causality and Controlling Disease is Based on Collaborative Research involving Multidisciplinary Approaches
    Skerratt, LF ; Garner, TWJ ; Hyatt, AD (SPRINGER, 2009-09-01)
    Understanding the causes of infectious disease to facilitate better control requires observational and experimental studies. Often these must be conducted at many scales such as at the molecular, cellular, organism, and population level. Studies need to consider both intrinsic and extrinsic factors affecting the pathogen/host interaction. They also require a combination of study methods covered by disciplines such as pathology, epidemiology, microbiology, and ecology. Therefore, it is important that disciplines work together when designing and conducting studies. Finally, we need to integrate and interpret data across levels and disciplines to better formulate control strategies. This requires another group of specialists with broad cross-disciplinary training in epidemiology and an ability to readily work with others.
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    Avian influenza at both ends of a migratory flyway: characterizing viral genomic diversity to optimize surveillance plans for North America
    Pearce, JM ; Ramey, AM ; Flint, PL ; Koehler, AV ; Fleskes, JP ; Franson, JC ; Hall, JS ; Derksen, DV ; Ip, HS (WILEY, 2009-11-01)
    Although continental populations of avian influenza viruses are genetically distinct, transcontinental reassortment in low pathogenic avian influenza (LPAI) viruses has been detected in migratory birds. Thus, genomic analyses of LPAI viruses could serve as an approach to prioritize species and regions targeted by North American surveillance activities for foreign origin highly pathogenic avian influenza (HPAI). To assess the applicability of this approach, we conducted a phylogenetic and population genetic analysis of 68 viral genomes isolated from the northern pintail (Anas acuta) at opposite ends of the Pacific migratory flyway in North America. We found limited evidence for Asian LPAI lineages on wintering areas used by northern pintails in California in contrast to a higher frequency on breeding locales of Alaska. Our results indicate that the number of Asian LPAI lineages observed in Alaskan northern pintails, and the nucleotide composition of LPAI lineages, is not maintained through fall migration. Accordingly, our data indicate that surveillance of Pacific Flyway northern pintails to detect foreign avian influenza viruses would be most effective in Alaska. North American surveillance plans could be optimized through an analysis of LPAI genomics from species that demonstrate evolutionary linkages with European or Asian lineages and in regions that have overlapping migratory flyways with areas of HPAI outbreaks.
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    Meeting report: Application of genotyping methods to assess risks from cryptosporidium in watersheds
    Ferguson, C ; Deere, D ; Sinclair, M ; Chalmers, RM ; Elwin, K ; Hadfield, S ; Xiao, LH ; Ryan, U ; Gasser, R ; El-Osta, YA ; Stevens, M (US DEPT HEALTH HUMAN SCIENCES PUBLIC HEALTH SCIENCE, 2006-03-01)
    A workshop titled "Application of Genotyping Methods to Assess Pathogen Risks from Cryptosporidium in Drinking Water Catchments" was held at the International Water Association biennial conference, Marrakech, Morocco, 23 September 2004. The workshop presented and discussed the findings of an interlaboratory trial that compared methods for genotyping Cryptosporidium oocysts isolated from feces. The primary goal of the trial and workshop was to assess the utility of current Cryptosporidium genotyping methods for determining the public health significance of oocysts isolated from feces in potable-water-supply watersheds. An expert panel of 16 watershed managers, public health practitioners, and molecular parasitologists was assembled for the workshop. A subordinate goal of the workshop was to educate watershed management and public health practitioners. An open invitation was extended to all conference delegates to attend the workshop, which drew approximately 50 interested delegates. In this report we summarize the peer consensus emerging from the workshop. Recommendations on the use of current methods by watershed managers and public health practitioners were proposed. Importantly, all the methods that were reported in the trial were mutually supporting and found to be valuable and worthy of further utility and development. Where there were choices as to which method to apply, the small-subunit ribosomal RNA gene was considered to be the optimum genetic locus to target. The single-strand conformational polymorphism method was considered potentially the most valuable for discriminating to the subtype level and where a large number of samples were to be analyzed. A research agenda for protozoan geneticists was proposed to improve the utility of methods into the future. Standardization of methods and nomenclature was promoted.
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    Isolation of a variant infectious bronchitis virus in Australia that further illustrates diversity among emerging strains
    Ignjatovic, J ; Gould, G ; Sapats, S (SPRINGER WIEN, 2006-08-01)
    Australian infectious bronchitis viruses (IBV) have undergone a separate evolution due to geographic isolation. Consequently, changes occurring in Australian IBV illustrate, independently from other countries, types of variability that could occur in emerging IBV strains. Previously, we have identified two distinct genetic groups of IBV, designated subgroups 1 and 2. IBV strains of subgroup 1 have S1 and N proteins that share a high degree of amino acid identity, 81 to 98% in S1 and 91 to 99% in N. Subgroup 2 strains possess S1 and N proteins that share a low level of identity with subgroup 1 strains: 54 to 62% in S1 and 60 to 62% in N. This paper describes the isolation and characterisation of a third, previously undetected genetic group of IBV in Australia. The subgroup 3 strains, represented by isolate chicken/Australia/N2/04, had an S1 protein that shared a low level of identity with both subgroups 1 and 2: 61 to 63% and 56 to 59%, respectively. However, the N protein and the 3' untranslated region were similar to subgroup 1: 90 to 97% identical with the N protein of subgroup 1 strains. This N4/02 subgroup 3 of IBV is reminiscent of two other strains, D1466 and DE072, isolated in the Netherlands and in the USA, respectively. The emergence of the subgroup 3 viruses in Australia, as well as the emergence of subgroup 2 in 1988, could not be explained by any of the mechanisms that are currently considered to be involved in generation of IBV variants.
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    Influence of muscle-tendon wrapping on calculations of joint reaction forces in the equine distal forelimb
    Merritt, JS ; Davies, HMS ; Burvill, C ; Pandy, MG (HINDAWI PUBLISHING CORPORATION, 2008-01-01)
    The equine distal forelimb is a common location of injuries related to mechanical overload. In this study, a two-dimensional model of the musculoskeletal system of the region was developed and applied to kinematic and kinetic data from walking and trotting horses. The forces in major tendons and joint reaction forces were calculated. The components of the joint reaction forces caused by wrapping of tendons around sesamoid bones were found to be of similar magnitude to the reaction forces between the long bones at each joint. This finding highlighted the importance of taking into account muscle-tendon wrapping when evaluating joint loading in the equine distal forelimb.
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    Flinders Island spotted fever rickettsioses caused by "marmionii strain of Rickettsia honei, eastern Australia
    Unsworth, NB ; Stenos, J ; Graves, SR ; Faa, AG ; Cox, GE ; Dyer, JR ; Boutlis, CS ; Lane, AM ; Shaw, MD ; Robson, J ; Nissen, MD (CENTERS DISEASE CONTROL & PREVENTION, 2007-04-01)
    Australia has 4 rickettsial diseases: murine typhus, Queensland tick typhus, Flinders Island spotted fever, and scrub typhus. We describe 7 cases of a rickettsiosis with an acute onset and symptoms of fever (100%), headache (71%), arthralgia (43%), myalgia (43%), cough (43%), maculopapular/petechial rash (43%), nausea (29%), pharyngitis (29%), lymphadenopathy (29%), and eschar (29%). Cases were most prevalent in autumn and from eastern Australia, including Queensland, Tasmania, and South Australia. One patient had a history of tick bite (Haemaphysalis novaeguineae). An isolate shared 99.2%, 99.8%, 99.8%, 99.9%, and 100% homology with the 17 kDa, ompA, gltA, 16S rRNA, and Sca4 genes, respectively, of Rickettsia honei. This Australian rickettsiosis has similar symptoms to Flinders Island spotted fever, and the strain is genetically related to R. honei. It has been designated the "marmionii" strain of R. honei, in honor of Australian physician and scientist Barrie Marmion.
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    Three rickettsioses, Darnley Island, Australia
    Unsworth, NB ; Stenos, J ; Faa, AG ; Graves, SR (CENTERS DISEASE CONTROL & PREVENTION, 2007-07-01)
    We report 3 rickettsioses on Darnley Island, Australia, in the Torres Strait. In addition to previously described cases of Flinders Island spotted fever (Rickettsia honei strain "marmionii"), we describe 1 case of Queensland tick typhus (R. australis) and 2 cases of scrub typhus caused by a unique strain (Orientia tsutsugamushi).
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    Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus
    Black, WD ; Hartley, CA ; Ficorilli, NP ; Studdert, MJ (SPRINGER WIEN, 2007-01-01)
    Equine rhinitis B virus (ERBV), genus Erbovirus, family Picornaviridae occurs as two serotypes, ERBV1 and ERBV2. An ERBV-specific nested reverse transcriptase-polymerase chain reaction (RT-PCR) that amplified a product within the 3D(pol) and 3' non-translated region of the viral genome was developed. The RT-PCR detected all 24 available ERBV1 isolates and one available ERBV2 isolate. The limit of detection for the prototype strain ERBV1.1436/71 was 0.1 50% tissue culture infectious doses. The RT-PCR was used to detect viral RNA in six of 17 nasopharyngeal swab samples from horses that had clinical signs of acute febrile respiratory disease but from which ERBV was not initially isolated in cell culture. The sequences of these six ERBV RT-PCR positive samples had 93-96% nucleotide identity with six other partially sequenced ERBV1 isolates and one ERBV2. ERBV was isolated from one of the six samples at fourth cell culture passage when it was shown that the addition of 20 mg/mL MgCl(2) to the cell culture medium enhanced the growth of the virus. This isolated virus was antigenically similar to ERBV2.313/75. Determination of the nucleotide sequence of the P1 region of the genome also indicated that the isolate was ERBV2, and it was therefore designated ERBV2.1576/99. This is the first reported isolation of ERBV in Australia. The study highlights the utility of PCR for the identification of viruses in clinical samples that may initially be considered negative by conventional cell culture isolation.
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    A New PCR-Based Approach Indicates the Range of Clonorchis sinensis Now Extends to Central Thailand
    Traub, RJ ; Macaranas, J ; Mungthin, M ; Leelayoova, S ; Cribb, T ; Murrell, KD ; Thompson, RCA ; Sripa, B (PUBLIC LIBRARY SCIENCE, 2009-01-01)
    Differentiation of the fish-borne trematodes belonging to the Opisthorchiidae, Heterophyidae and Lecithodendriidae is important from a clinical and epidemiological perspective, yet it is impossible to do using conventional coprological techniques, as the eggs are morphologically similar. Epidemiological investigation therefore currently relies on morphological examination of adult worms following expulsion chemotherapy. A PCR test capable of amplifying a segment of the internal transcribed spacer region of ribosomal DNA for the opisthorchiid and heterophyid flukes eggs taken directly from faeces was developed and evaluated in a rural community in central Thailand. The lowest quantity of DNA that could be amplified from individual adults of Opisthorchis viverrini, Clonorchis sinensis and Haplorchis taichui was estimated at 0.6 pg, 0.8 pg and 3 pg, respectively. The PCR was capable of detecting mixed infection with the aforementioned species of flukes under experimental conditions. A total of 11.6% of individuals in rural communities in Sanamchaikaet district, central Thailand, were positive for 'Opisthorchis-like' eggs in their faeces using conventional parasitological detection techniques. In comparison to microscopy, the PCR yielded a sensitivity and specificity of 71.0% and 76.7%, respectively. Analysis of the microscopy-positive PCR products revealed 64% and 23% of individuals to be infected with O. viverrini and C. sinensis, respectively. The remaining 13% (three individuals) were identified as eggs of Didymozoidae, presumably being passed mechanically in the faeces following the ingestion of infected fishes. An immediate finding of this study is the identification and first report of a C. sinensis-endemic community in central Thailand. This extends the known range of this liver fluke in Southeast Asia. The PCR developed herein provides an important tool for the specific identification of liver and intestinal fluke species for future epidemiological surveys.