Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus.
Johnny D Callahan, Fred Brown, Fernando A Osorio, Jung H Sur, Ed Kramer, Gary W Long, Juan Lubroth, Stefanie J Ellis, Katina S Shoulars, Kristin L Gaffney, Daniel L Rock, William M Nelson
Tetracore Inc, Gaithersburg, MD 20878, USA.
OBJECTIVE To evaluate a portable real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay designed to detect all 7 viral serotypes of foot-and-mouth disease virus (FMDV). DESIGN Laboratory and animal studies. STUDY POPULATION Viruses grown in tissue culture and animals experimentally infected with FMDV. PROCEDURE 1 steer, pig, and sheep were infected with serotype O FMDV. Twenty-four hours later, animals were placed in separate rooms that contained 4 FMDV-free, healthy animals of the same species. Oral and nasal swab specimens, oropharyngeal specimens obtained with a probang, and blood samples were obtained at frequent intervals, and animals were observed for fever and clinical signs of foot-and-mouth disease (FMD). Samples from animals and tissue cultures were assayed for infectious virus and viral RNA. RESULTS The assay detected viral RNA representing all 7 FMDV serotypes grown in tissue culture but did not amplify a panel of selected viruses that included those that cause vesicular diseases similar to FMD; thus, the assay had a specificity of 100%, depending on the panel selected. The assay also met or exceeded sensitivity of viral culture on samples from experimentally infected animals. In many instances, the assay detected viral RNA in the mouth and nose 24 to 96 hours before the onset of clinical disease. CONCLUSIONS AND CLINICAL RELEVANCE The assay reagents are produced in a vitrified form, which permits storage and transportation at ambient temperatures. The test can be performed in 2 hours or less on a portable instrument, thus providing a rapid, portable, sensitive, and specific method for detection of FMDV.
Vet Med Int. 2011 ;2011 :905768 21776357
A Brief Review on Diagnosis of Foot-and-Mouth Disease of Livestock: Conventional to Molecular Tools.
Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati 781022, India.
Foot-and-mouth disease (FMD) is one of the highly contagious diseases of domestic animals. Effective control of this disease needs sensitive, specific, and quick diagnostic tools at each tier of control strategy. In this paper we have outlined various diagnostic approaches from old to new generation in a nutshell. Presently FMD diagnosis is being carried out using techniques such as Virus Isolation (VI), Sandwich-ELISA (S-ELISA), Liquid-Phase Blocking ELISA (LPBE), Multiplex-PCR (m-PCR), and indirect ELISA (DIVA), and real time-PCR can be used for detection of antibody against nonstructural proteins. Nucleotide sequencing for serotyping, microarray as well as recombinant antigen-based detection, biosensor, phage display, and nucleic-acid-based diagnostic are on the way for rapid and specific detection of FMDV. Various pen side tests, namely, lateral flow, RT-LAMP, Immunostrip tests, and so forth. are also developed for detection of the virus in field condition.
Department of Mathematics and Statistics, Coastal Carolina University, Conway, SC 29528, USA.
Monitoring populations of hosts as well as insect vectors is an important part of agricultural and public health risk assessment. In applications where pathogen prevalence is likely low, it is common to test pools of subjects for the presence of infection, rather than to test subjects individually. This technique is known as pooled (group) testing. In this paper, we revisit the problem of estimating the population prevalence p from pooled testing, but we consider applications where inverse binomial sampling is used. Our work is unlike previous research in pooled testing, which has largely assumed a binomial model. Inverse sampling is natural to implement when there is a need to report estimates early on in the data collection process and has been used in individual testing applications when disease incidence is low. We consider point and interval estimation procedures for p in this new pooled testing setting, and we use example data sets from the literature to describe and to illustrate our methods.
Virol J. 2011 Mar 31;8 (1):148 21453461
A highly sensitive detection for foot-and-mouth disease virus by gold nanopariticle improved immuno-PCR.
ABSTRACT: BACKGROUND: Foot-and-mouth disease (FMD) is one of the most contagious of all artiodactyl animal diseases, and its infection has an obvious ability to spread over long distances and to contribute to epidemics in FMD-free areas. A highly sensitive and specfic method is required to detect FMDV. In this study, we evaluated the usefulness of a bio-barcode assay (BCA) technique for detecting clinical samples of FMDV. METHODS: Highly sensitive gold nanopariticle (GNP) improved immuno -PCR (GNP-IPCR) which derived from the bio-barcode assay (BCA) was designed for the detection of FMDV. The target viral particles were captured by a polyclonal antibody coated on ELISA microplate, followed by adding GNP which was dually modified with oligonucleotides and a FMDV specific monoclonal anibody (MAb) 1D11 to form a sandwiched immune complex. After the formation of immuno-complex, the signal DNA was released by heating, and consequently characterized by PCR and real time PCR. RESULTS: The detection limit of GNP-PCR could reach to 10 fg/ml purified FMDV particles, and the assay can detect clinical samples of FMDV with highly sensitivity, while detect limit of conventional ELISA is 100 ng/ml in this study. CONCLUSION: GNP-IPCR may provide a highly senstive method for the detection of FMDV.
Rescue of foot-and-mouth disease viruses that are pathogenic for cattle from preserved viral RNA samples.
National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark. firstname.lastname@example.org
Foot and mouth disease is an economically important disease of cloven-hoofed animals including cattle, sheep and pigs. It is caused by a picornavirus, foot-and-mouth disease virus (FMDV), which has a positive sense RNA genome which, when introduced into cells, can initiate virus replication. A system has been developed to rescue infectious FMDV from RNA preparations generated from clinical samples obtained under experimental conditions and then applied to samples collected in the "field". Clinical samples from suspect cases of foot-and-mouth disease (FMD) were obtained from within Pakistan and Afghanistan. The samples were treated to preserve the RNA and then transported to National Veterinary Institute, Lindholm, Denmark. Following RNA extraction, FMDV RNA was quantified by real-time RT-PCR and samples containing significant levels of FMDV RNA were introduced into susceptible cells using electroporation. Progeny viruses were amplified in primary bovine thyroid cells and characterized using antigen ELISA and also by RT-PCR plus sequencing. FMD viruses of three different serotypes and multiple lineages have been successfully rescued from the RNA samples. Two of the rescued viruses (of serotype O and Asia 1) were inoculated into bull calves under high containment conditions. Acute clinical disease was observed in each case which spread rapidly from the inoculated calves to in-contact animals. Thus the rescued viruses were highly pathogenic. The availability of the rescued viruses enabled serotyping by antigen ELISA and facilitated genome sequencing. The procedure described here should improve the characterization of FMDVs circulating in countries where the disease is endemic and thus enhance disease control globally.
Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing.
Caroline F Wright, Marco J Morelli, Gaël Thébaud, Nick J Knowles, Pawel Herzyk, David J Paton, Daniel T Haydon, Donald P King
University of Glasgow Centre for Virus Research, Glasgow G12 8QQ, UK.
The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10(-4) per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.
Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam in 2007.
S Metwally, F Mohamed, K Faaberg, T Burrage, M Prarat, K Moran, A Bracht, G Mayr, M Berninger, L Koster, T L To, V L Nguyen, M Reising, J Landgraf, L Cox, J Lubroth, C Carrillo
FAO Reference Center for Vesicular Diseases, USDA, APHIS, Foreign Animal Disease Diagnostic Laboratory, National Veterinary Service Laboratories (NVSL), Plum Island Animal Disease Center (PIADC), Greenport, NY, USA. Samia.A.Metwally@aphis.usda.gov
In 2007, Vietnam experienced swine disease outbreaks causing clinical signs similar to the 'porcine high fever disease' that occurred in China during 2006. Analysis of diagnostic samples from the disease outbreaks in Vietnam identified porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV-2). Additionally, Escherichia coli and Streptococcus equi subspecies zooepidemicus were cultured from lung and spleen, and Streptococcus suis from one spleen sample. Genetic characterization of the Vietnamese PRRSV isolates revealed that this virus belongs to the North American genotype (type 2) with a high nucleotide identity to the recently reported Chinese strains. Amino acid sequence in the nsp2 region revealed 95.7-99.4% identity to Chinese strain HUN4, 68-69% identity to strain VR-2332 and 58-59% identity to strain MN184. A partial deletion in the nsp2 gene was detected; however, this deletion did not appear to enhance the virus pathogenicity in the inoculated pigs. Animal inoculation studies were conducted to determine the pathogenicity of PRRSV and to identify other possible agents present in the original specimens. Pigs inoculated with PRRSV alone and their contacts showed persistent fever, and two of five pigs developed cough, neurological signs and swollen joints. Necropsy examination showed mild to moderate bronchopneumonia, enlarged lymph nodes, fibrinous pericarditis and polyarthritis. PRRSV was re-isolated from blood and tissues of the inoculated and contact pigs. Pigs inoculated with lung and spleen tissue homogenates from sick pigs from Vietnam developed high fever, septicaemia, and died acutely within 72 h, while their contact pigs showed no clinical signs throughout the experiment. Streptococcus equi subspecies zooepidemicus was cultured, and PRRSV was re-isolated only from the inoculated pigs. Results suggest that the cause of the swine deaths in Vietnam is a multifactorial syndrome with PRRSV as a major factor.
The early pathogenesis of foot-and-mouth disease in cattle after aerosol inoculation. Identification of the nasopharynx as the primary site of infection.
Foreign Animal Disease Research Unit, USDA/ARS Plum Island Animal Disease Center, PO Box 848, Greenport, NY 11944, USA. email@example.com
To characterize the early events of foot-and-mouth disease virus (FMDV) infection in cattle subsequent to simulated natural exposure, 16 steers were aerosol inoculated with FMDV and euthanized at various times. Samples were collected from each steer antemortem (serum, nasal swabs, and oral swabs) and postmortem (up to 40 tissues per animal) and screened for FMDV by virus isolation and for FMDV RNA by real-time reverse transcription polymerase chain reaction. Tissues that tested positive for FMDV or viral RNA were examined by immunohistochemistry and multichannel immunofluorescence microscopy. In previremic steers, FMDV was most consistently localized to nasopharyngeal tissues, thereby indicating this region as the most important site of primary viral replication. The earliest site of microscopic localization of FMDV antigens was the lymphoid follicle-associated epithelium of the pharyngeal mucosa-associated lymphoid tissue of the nasopharynx at 6 hours postaerosolization. At early time points after aerosol inoculation, viral antigens colocalized with cytokeratin-positive pharyngeal epithelial cells; intraepithelial FMDV-negative, MHCII/CD11c-double-positive dendritic cells were present in close proximity to FMDV-positive cells. Onset of viremia coincided with marked increase of viral loads in pulmonary tissues and with substantial decrease of viral detection in nasopharyngeal tissues. These data indicate that subsequent to aerogenous exposure to FMDV, the temporally defined critical pathogenesis events involve (1) primary replication in epithelial cells of the pharyngeal mucosa-associated lymphoid tissue crypts and (2) subsequent widespread replication in pneumocytes in the lungs, which coincides with (3) the establishment of sustained viremia.
Virology. 2010 Sep 15;405 (1):149-56 20580394
Domain disruptions of individual 3B proteins of foot-and-mouth disease virus do not alter growth in cell culture or virulence in cattle.
Juan M Pacheco, Maria E Piccone, Elizabeth Rieder, Steven J Pauszek, Manuel V Borca, Luis L Rodriguez
Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York, USA.
Picornavirus RNA replication is initiated by a small viral protein primer, 3B (also known as VPg), that is covalently linked to the 5' terminus of the viral genome. In contrast to other picornaviruses that encode a single copy of 3B, foot-and-mouth disease virus (FMDV) encodes three copies of 3B. Viruses containing disrupted native sequence or deletion of one of their three 3B proteins were derived from a FMDV A24 Cruzeiro full-length cDNA infectious clone. Mutant viruses had growth characteristics similar to the parental virus in cells. RNA synthesis and protein cleavage processes were not significantly affected in these mutant viruses. Cattle infected by aerosol exposure with mutant viruses developed clinical disease similar to that caused by the parental A24 Cruzeiro. Therefore, severe domain disruption or deletion of individual 3B proteins in FMDV do not affect the virus' ability to replicate in vitro and cause clinical disease in cattle.
Scott M Reid, Kenneth E Pierce, Rohit Mistry, Sukvinder Bharya, Juliet P Dukes, Carmelo Volpe, Lawrence J Wangh, Donald P King
Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, UK.
A reverse transcription Linear-After-The-Exponential polymerase chain reaction (RT LATE-PCR) assay was evaluated for detection of foot-and-mouth disease virus (FMDV). This pan-serotypic assay targets highly conserved sequences within the 3D (RNA polymerase) region of the FMDV genome, and uses end-point hybridisation analysis of a single mismatch-tolerant low temperature probe to confirm the identity of the amplicons. An Armored RNA served as an internal control to validate virus negative results. The ability of the assay to identify FMDV was directly compared to a real-time RT-PCR assay routinely used by reference laboratories. The analytical sensitivity of the RT LATE-PCR assay was 10 genomic copies and the dynamic range of the test was identical to real-time RT-PCR based on decimal dilutions of an FMDV-positive sample. This pan-serotypic assay was able to detect FMDV in a broad range of clinical samples collected from field cases of FMD (n = 121), while samples of other viruses causing vesicular disease in livestock and genetic relatives of FMDV were negative. In addition to the laboratory-based utility of this diagnostic test, the RT LATE-PCR assay format has potential application for use in a portable ("point-of-care") device designed to achieve rapid detection of FMDV in the field.
A one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe.
Guoyuan Wen, Jun Yang, Qingping Luo, Zhibin Hu, Nianhua Song, Rongrong Zhang, Hongling Wang, Diyun Ai, Ling Luo, Huabin Shao
Institute of Animal Husbandry and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan, 430070, China.
The aim of this study was to develop a one-step real-time reverse transcription-polymerase chain reaction assay using the minor groove binding probe (MGB rRT-PCR) for rapid and quantitative detection of classical swine fever virus (CSFV). The method, which targets the 5'-nontranslated region (5'NTR) of the viral genome, detected all CSFV isolate tested, but not heterologous pathogens. Using an in vitro transcript of the 5'NTR as a quantitative standard for the CSFV genome copy number, the assay had a detection limit of 10 copies/reaction, and the standard curve had a linear range from 10 to 10(7) copies/reaction, with good reproducibility. As determined by an end-point dilution comparison, in most case, the sensitivity of the MGB rRT-PCR was approximately 10-fold higher than that of virus isolation and the rRT-PCR using the standard Taqman probe (standard rRT-PCR). The agreement between the MGB rRT-PCR and standard rRT-PCR, or virus isolation was 93.3% and 76.7%, respectively, when detecting 261 field samples. Due to its rapidity, high specificity and sensitivity, the MGB rRT-PCR assay provides a valuable tool for diagnosis and molecular studies of CSFV biology.
Other papers by authors:
A synthetic peptide containing the consensus sequence of the G-H loop region of foot-and-mouth disease virus type-O VP1 and a promiscuous T-helper epitope induces peptide-specific antibodies but fails to protect cattle against viral challenge.
Agricultural Research Service, US Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY 11944, USA. firstname.lastname@example.org
A pilot study was carried out in cattle to determine the immunogenicity of a synthetic consensus peptide comprising the G-H loop region of foot-and-mouth disease virus (FMDV) type-O VP1 and a non-VP1 T-helper (Th) epitope. Cattle vaccinated intramuscularly either once (n = 5) or twice (n = 4) with 50 microg of the peptide preparation at a 21-day interval developed antibodies to the peptide as determined by ELISA with the exception of one steer that received a single dose. However, neutralizing antibody titers against FMDV type-O were modest and all animals presented with clinical FMD signs upon challenge 21 days after the last vaccination. In contrast, four of the five animals inoculated with an inactivated FMD type-O commercially prepared vaccine developed neutralizing antibodies and were fully protected against clinical disease following virus challenge 21 days post-vaccination (dpv). Nucleotide sequence comparison of the VP1 region between the challenge virus and RT-PCR products recovered from a lesion of the peptide-vaccinated animal with the highest neutralizing antibody titer 5 days post-challenge (dpc) showed no evidence for selection of a neutralization-resistant mutant. We conclude that although the synthetic peptide induced an antibody response in cattle, it failed to confer protection against FMDV challenge.
Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time reverse transcription-polymerase chain reaction assays.
Donald P King, Nigel P Ferris, Andrew E Shaw, Scott M Reid, Geoff H Hutchings, Angelica C Giuffre, John M Robida, Johnny D Callahan, William M Nelson, Tammy R Beckham
Institute for Animal Health, Pirbright Laboratory, Surrey, UK. email@example.com
Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5' untranslated region (5'UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1%(347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5'UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.
A peptide vaccine administered transcutaneously together with cholera toxin elicits potent neutralising anti-FMDV antibody responses.
Anne-Sophie Beignon, Fred Brown, Pierre Eftekhari, Ed Kramer, Jean-Paul Briand, Sylviane Muller, Charalambos D Partidos
UPR 9021, Institut de Biologie Moléculaire et Cellulaire, CNRS, 15 rue René Descartes, F-67084 Strasbourg, France.
In this study a synthetic peptide representing residues 141-159 from the GH loop of VP1 protein of foot-and-mouth disease virus was tested for its capacity to elicit virus neutralising antibodies in mice after transcutaneous immunisation. Topical application of the peptide conjugated to bovine serum albumin together with cholera toxin as an adjuvant elicited anti-peptide antibody responses with strong virus neutralising activity. The combination of cholera toxin with an immunostimulatory CpG motif resulted in the induction of IgG1 and IgG2a anti-peptide antibodies with significantly enhanced virus neutralising activity. To shed more light on the mechanisms of cholera toxin adjuvanticity we demonstrated its binding to keratinocytes via GM(1)-gangliosides. This was followed by an increase of the intracellular cAMP and the rapid diffusion of cholera toxin throughout the epidermis. These findings demonstrate that peptide-based vaccines when combined with the appropriate adjuvant(s) can elicit potent virus neutralising antibody responses after transcutaneous immunisation. However, experiments in target species will be required to confirm the potential of this simple vaccination procedure in livestock.
Immunization with peptide-functionalized carbon nanotubes enhances virus-specific neutralizing antibody responses.
Davide Pantarotto, Charalambos D Partidos, Johan Hoebeke, Fred Brown, Ed Kramer, Jean-Paul Briand, Sylviane Muller, Maurizio Prato, Alberto Bianco
Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, Immunologie et Chimie Thérapeutiques, 15 Rue René Descartes, 67084 Strasbourg, France.
Thomas Mather, Tsutomu Takeda, Jodie Tassello, Asa Ohagen, Diana Serebryanik, Ed Kramer, Fred Brown, Robert Tesh, Bernadette Alford, John Chapman, Aris Lazo
V.I. Technologies, Inc., Watertown, Massachusetts 02472, USA.
BACKGROUND The outbreak of West Nile virus (WNV) is the most recent reminder that the blood supply continues to be vulnerable to emerging and reemerging pathogens. A potentially prospective approach to reducing the risk of transfusion-transmitted infections of a known or newly emerging microbe is implementation of a broad-spectrum pathogen reduction technology. The purpose of this study was to evaluate the susceptibility of WNV to PEN110 inactivation in RBCs and to characterize the WNV interaction with blood, including the stability of WNV in RBCs stored at 1 to 6 degrees C, its distribution and infectivity, and its ability to infect WBCs. STUDY DESIGN AND METHODS Inactivation was performed with three WNV isolates spiked into WBC-reduced RBCs. The stability of the virus was evaluated by spiking two viral loads into RBCs followed by storing at 1 to 6 degrees C for up to 42 days. The distribution of the virus in plasma, RBCs, and PBMCs was evaluated with whole blood from infected hamsters. Finally, in vitro propagation of WNV was evaluated with the THP-1 cell line and primary monocytes. RESULTS The kinetics of PEN110 inactivation of WNV isolates RI-44, NJ-176, and 99-3494031 were fast and complete within 24 hours with reduction factors of 5 to 7 log plaque-forming units per mL. WNV remained infectious for up to 42 days at 1 to 6 degrees C. The WNV titers in whole blood, plasma, RBCs, and PBMC fractions were equally distributed and ranged from 2 to 3 log tissue culture infectious dose 50 percent per mL. Productive infection of stimulated monocytes and THP-1 cells was also demonstrated. CONCLUSIONS These studies demonstrated that PEN110 efficiently inactivated WNV in RBCs and whole blood from infected hamsters to the limit of detection. WNV survived in RBCs stored at 1 to 6 degrees C with a gradual loss of titer but infectivity could still be observed for up to 42 days. In addition, it was observed that WNV was equally distributed in all blood fractions including PBMCs and it was possible to establish productive infection of a human monocytic cell line and stimulated human monocytes.
Applying peptide antigens onto bare skin: induction of humoral and cellular immune responses and potential for vaccination.
Charalambos D Partidos, Anne-Sophie Beignon, Fred Brown, Ed Kramer, Jean-Paul Briand, Sylviane Muller
UPR 9021, Institut de Biologie Moléculaire et Cellulaire, CNRS, 15 Rue René Descartes, F-67084, Strasbourg, France. H.firstname.lastname@example.org
The development of non-invasive immunisation procedures is a top priority for public health agencies when it is realised that the current immunisation practices are unsafe, particularly in developing countries due to the widespread reuse of non-sterile syringes. There is a risk of abscess formation resulting in impairment of meat quality or the value of the hide, and the risk of transmission of infectious diseases when vaccines are administered to food animals by injection. Recently, the skin has emerged as an alternative route for non-invasive delivery of vaccines. Topical application of various types of antigens (mainly proteins and toxoids) with an adjuvant resulted in the induction of systemic and mucosal immune responses. However, due to skin barrier constraints and the physicochemical properties of large molecular weight proteins, the immune responses are variable and require further optimisation. Small molecular size synthetic peptides when applied onto bare skin with an adjuvant are effective immunogens, inducing both humoral and cellular immune responses. Their use as vaccines offers considerable advantages over conventional preparations in terms of safety, purity, stability, availability and cost. Therefore, they could be the most suitable candidate immunogens for skin immunisation. This review describes our recent observations on the immunogenicity of synthetic peptides applied onto bare skin in relation to vaccination.
Chang Yi Wang, Tseng Yuan Chang, Alan M Walfield, John Ye, Ming Shen, Shih Ping Chen, Ming Chang Li, Yeou Liang Lin, Ming Hwa Jong, Ping Cheng Yang, Nancy Chyr, Ed Kramer, Fred Brown
United Biomedical Inc., 25 Davids Drive, Hauppauge, NY 11788, USA. email@example.com
We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G-H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G-H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 microg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.
BMC Vet Res. 2012 Nov 23;8 (1):229 23174032
Wei Li, Zhangyong Ning, Wenbo Hao, Deguang Song, Feng Gao, Kui Zhao, Xiaoqing Liao, Ming Li, Daniel L Rock, Shuhong Luo
ABSTRACT: BACKGROUND: Orf is a zoonotic and epitheliotrophic contagious disease that mainly affects sheep, goats, wild ruminants, and humans with a worldwide distribution. To date, there is little information on the characterization of ORFV strains that are endemic in Mainland China. In addition, the relationship between the severity of disease and the molecular profile of ORFV strains has not been fully elucidated. RESULTS: From the recent outbreak of a sheep herd in Nongan, northeast of China, the novel orf virus (ORFV) strain NA1/11 was successfully isolated. Western blot analysis indicated that the NA1/11 strain cross reacts with monoclonal antibody A3 and infected sheep ORFV antiserum. The purified virions revealed the typical ovoid shape when observed by atomic force microscopy. To determine the genetic characteristics of the NA1/11 strain, the sequences of ORFV011 (B2L), ORFV059 (F1L), ORFV109, ORFV110 and ORFv132 (VEGF) genes were amplified and compared with reference parapoxvirus strains. Non-metric multidimensional scaling (nMDS) was performed to analyze the nucleotide similarities between different ORFV strains. CONCLUSIONS: Phylogenetic analysis based on ORFV 011 nucleotide sequences showed that the NA1/11strain was closely related to Xinjiang and Gansu strains. ORFV110 and ORFV132 genes are highly variable. The results revealed that precise phylogenetic analysis might provide evidence for genetic variation and movement of circulating ORFV strains in Northeast China. In addition, NMDS analysis showed that geographic isolation and animal host are likely major factors resulting in genetic differences between ORFV strains.
Virology. 2012 Sep 17;: 22995188
Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1.
Lalit K Beura, Sakthivel Subramaniam, Hiep L X Vu, Byungjoon Kwon, Asit K Pattnaik, Fernando A Osorio
School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900, USA.
The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFN-suppression. A mutant virus (16-5A, in which residues 16-20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-5A virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs.
Animal Health Service, Food and Agriculture Organization, Viale delle Terme di Caracalla, 00153, Rome, Italy, Juan.Lubroth@fao.org.
The Food and Agriculture Organization (FAO) of the United Nation's view on One Health is broad as it extends from human, animal-domestic and wildlife-and environmental health. Though the nidus of work originated within FAO's animal health service of the Agriculture and Consumer Protection Department, it is clearly an area of work that would include other departments such as Natural Resources Management and the Environment, Forestry, Fisheries and Aquaculture, Economic and Social Development, Legal Services, and communication. In terms of risk assessment and risk mitigation to health threats at the human-animal-ecosystem interface FAO works closely with its global partners, World Health Organisation and the World Organisation for animal health (the "Tripartite"). FAO's animal health service sees its work in One Health as contributing to all eight Millennium Development Goals, recognising the importance of animal health to human health, food safety, nutrition and food security, ameliorating poverty and hunger, natural resource management and partnerships. Some examples of FAO's operationalising One Health approaches or principles are introduced.
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J Vet Med Sci. 2012 Sep 5;: 22971595
Simultaneous Detection of Canine Respiratory Disease Associated Viruses by a Multiplex Reverse Transcription-Polymerase Chain Reaction Assay.
Animal, Plant and Fisheries Quarantine and Inspection Agency.
A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the simultaneous detection of canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), and canine influenza virus (CIV). These viral pathogens are all causative agents of canine infectious respiratory disease (CIRD). The sensitivity and specificity of the mRT-PCR were determined by comparing it to a rapid antigen test (RAT) or immuno-chromatography test kit and reverse transcription-polymerase chain reaction (RT-PCR) in the detection of CDV, CRCoV, and CIV antigens present in 100 clinical samples (nasal swabs and whole blood samples) from 50 dogs with respiratory disease symptoms. This study revealed that mRT-PCR had almost exactly the same performance or results were almost 100% in agreement with that of RT-PCR and RAT both in terms of the assay sensitivity and specificity which was more highly evident in detecting CIV, CDV, and CRCoV antigens present in canine nasal swab samples. Therefore, this assay could be a better alternative for the definitive and simultaneous ante-mortem detection of the three viral pathogens that cause CIRD by using nasal swabs.
Vet J. 2012 Mar 23;: 22445313
Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL, Utrecht, The Netherlands.
A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.
Vet J. 2012 Feb 17;: 22342891
Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences.
Foreign Animal Disease Research Unit, Agriculture Research Service, US Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY 11944, USA.
A novel direct contact transmission model for the study of foot-and-mouth disease virus (FMDV) infection of swine was utilized to investigate transmission characteristics of three FMDV strains belonging to serotypes A, O and Asia1. Each strain demonstrated distinct transmission characteristics and required different exposure times to achieve successful contact transmission. While a 4h exposure was sufficient for strain A24 Cruzeiro (A24Cru), both O1 Manisa and Asia1 Shamir transmission required 18h or more. Viral excretion levels from donors (for all three strains) and virus present in room air (for A24Cru and O1 Manisa) were evaluated and associated with clinical signs and observed transmission pattern. Although all directly inoculated donor animals showed acute FMD, A24Cru had the highest levels of viral shedding in saliva and nasal swabs followed by O1 Manisa and Asia1 Shamir. Virus levels in room air were higher and were detected longer for A24Cru than for O1 Manisa. These results provide direct evidence for important strain-specific variation in transmission characteristics and emphasize the need for thorough evaluation of different FMDV viral strains using a well defined contact transmission methodology. This information is critical for vaccine and biotherapeutic efficacy testing, pathogenesis and disease modeling of FMDV transmission.
Transbound Emerg Dis. 2011 Dec 28;: 22212855
Detection and genetic characterization of foot-and-mouth disease viruses in samples from clinically healthy animals in endemic settings.
S M Jamal, G Ferrari, M Hussain, A H Nawroz, A A Aslami, E Khan, S Murvatulloev, S Ahmed, G J Belsham
National Veterinary Laboratory, Islamabad, Pakistan Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark Food and Agriculture Organization of the United Nations, Rome, Italy FAO Project, GTFS//INT/907/ITA, National Veterinary Laboratory, Islamabad, Pakistan FAO Project, GTFS//INT/907/ITA, Kabul, Afghanistan FAO Project, GTFS//INT/907/ITA, Dushanbe, Tajikistan.
A total of 1501 oral swab samples from Pakistan, Afghanistan and Tajikistan were collected from clinically healthy animals between July 2008 and August 2009 and assayed for the presence of foot-and-mouth disease virus (FMDV) RNA. The oral swab samples from two (of four) live animal markets in Pakistan (n = 245), one (of three) live animal market in Afghanistan (n = 61) and both the live animal markets in Tajikistan (n = 120) all tested negative. However, 2 of 129 (∼2%) samples from Gondal and 11 of 123 (9%) from Chichawatni markets in Pakistan were positive for FMDV RNA. Similarly, 12 of 81 (15%) samples from Kabul and 10 of 20 (50%) from Badakhshan in Afghanistan were found to be positive. Serotypes A and O of FMDV were identified within these samples. Oral swab samples were also collected from dairy colonies in Harbanspura, Lahore (n = 232) and Nagori, Karachi (n = 136), but all tested negative for FMDV. In the Landhi dairy colony, Pakistan, a cohort of 179 apparently healthy animals was studied. On their arrival within the colony, thirty-nine (22%) of these animals were found positive for FMDV RNA (serotype A was identified), while 130 (72.6%) had antibodies to FMDV non-structural proteins. Thus, newly introduced animals may be a significant source of the disease in the colony. Only two animals from the cohort were detected as becoming positive for FMDV RNA during a follow-up period of 4 months; however, only 10 animals remained negative for anti-NSP antibodies during this period.
Masahiro Miyoshi, Shima Yoshizumi, Setsuko Ishida, Rika Komagome, Hideki Nagano, Shinichi Kudo, Motohiko Okano
Center for Infectious Diseases Control, Hokkaido Institute of Public Health, North 19 West 12, Kita-ku, Sapporo 060-0819, Japan. firstname.lastname@example.org
The rapid determination system of viral genome sequences (the RDV method) consists of detecting and determining the nucleotide sequences of viral genomes without using specific primers. To evaluate the usefulness of the RDV method, the detection of human norovirus (NV) genomes in stool specimens was investigated. In addition, the effect of nuclease treatment of the process was examined. A total of 23 human stool specimens were used, all of which were collected from patients with acute viral gastroenteritis, and were shown to contain NV genomes and also determined the cDNA copy numbers by the real-time reverse transcriptase-polymerase chain reaction. NV genomes were detected by the RDV method with nuclease treatment in nine specimens containing cDNA copies ranging between 6.2×10(9) and 9.8×10(11)/g stool. In contrast, NV genome was found by the method in 15 specimens without nuclease treatment and the number of NV cDNA copies ranged between 1.2×10(6) and 9.8×10(11)/g stool. These results suggest that the RDV method has potential for detecting viral genomes in stool specimens. The procedure without a step of nuclease treatment appears to be sensitive.
Serotype Identification and VP1 Coding Sequence Analysis of Foot-and-Mouth Disease Viruses from Outbreaks in Eastern and Northern Uganda in 2008/9.
Molecular Biology Laboratory, Institute of Environment and Natural Resources, Makerere University, Kampala, Uganda National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark National Animal Disease Diagnostics and Epidemiology Centre, Ministry of Agriculture Animal Industry and Fisheries, Entebbe, Uganda.
In April 2008, foot-and-mouth disease (FMD) outbreaks were reported in Kamuli district of the eastern region of Uganda. Soon after lifting the quarantines in this area, further FMD outbreaks were reported in northern Uganda, which spread to more than 10 districts. The aim of this study was to identify the serotype and compare the variable protein (VP)1 coding sequences of the viruses responsible for FMD outbreaks during 2008 and 2009, to trace the transmission pathways of the disease in Uganda. Probang and epithelial swab samples were collected from cattle with clinical signs of FMD in the two regions, and the presence of FMDV RNA in these samples was determined using a standard diagnostic RT-PCR assay. From the total of 27 positive samples, the VP1 coding region was amplified and sequenced. Each of these sequences showed >99% identity to each other, and just five distinct sequences were identified. BLAST searches and phylogenetic analysis of the complete variable protein (VP)1 coding sequences revealed that they belonged to serotype O, topotype EA-2. The close similarity between the virus sequences suggested introduction from a single source. We therefore conclude that FMD in the northern region of Uganda was most likely introduced from the outbreak in the eastern region across Lake Kyoga through movement of live animals. This has significant implications for the effectiveness of the current FMD control measures.
Vet J. 2011 Nov 22;: 22115952
Rapid detection of foot-and-mouth disease virus using a field-portable nucleic acid extraction and real-time PCR amplification platform.
Mikidache Madi, Andrea Hamilton, David Squirrell, Valerie Mioulet, Phillip Evans, Martin Lee, Donald P King
Institute for Animal Health, Ash Road, Pirbright GU24 0NF, UK.
Rapid and accurate field diagnostic tools can be used to support clinical diagnosis during outbreaks of exotic livestock diseases. This study evaluated a mobile PCR amplification platform that performs RNA extraction, real-time reverse-transcription PCR (rRT-PCR) and interpretation of results without operator intervention. Initial studies showed that there was equivalence between the detection limit generated by RNA extracted using the mobile platform and an automated laboratory-based system. In subsequent studies, two validated laboratory-based foot-and-mouth disease virus (FMDV)-specific rRT-PCRs were transferred onto the mobile platform and all assay steps (RT incubation and PCR amplification) were performed with non-lyophilised reagents using an optimised protocol of less than 60min. The limit of detection of the rRT-PCR on the mobile PCR platform was equivalent to an automated laboratory-based assay used for routine diagnosis of FMDV and there was concordance between these methods for results generated using samples in a reference laboratory proficiency panel. Future studies will be directed at the development and validation of commercially-viable consumables using lyophilised PCR reagents for FMDV and the evaluation of this technology in FMD endemic countries using field samples.
Virol J. 2011 ;8 :464 21978536
Establishing a TaqMan-based real-time PCR assay for the rapid detection and quantification of the newly emerged duck Tembusu virus.
Division of Avian Infectious Diseases, Key Laboratory of Animal Parasitology of the Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Minhang District, Shanghai 200241, China.
To establish an accurate, rapid, and a quantifiable method for the detection of the newly emerged duck Tembusu virus (DTMUV) that recently caused a widespread infectious disease in ducks in China, we developed a TaqMan-based real-time PCR assay by using E gene-specific primers and a TaqMan probe. This real-time PCR assay was 100 times more sensitive than the conventional PCR. The reproducibility and specificity of the real-time PCR assay were confirmed using plasmids containing E genes or RNAs and DNAs extracted from well-known viruses causing duck diseases. The reliability of this real-time PCR assay was confirmed in 19 of the 24 swab samples, 22 of the 24 tissue samples collected from experimentally infected ducks, as well as 15 of the 21 clinical samples collected from sick ducks since they were verified as DTMUV-positive. The results reveal that the newly established real-time PCR assay might be a useful diagnostic method for epidemiologically investigating and closely observing the newly emerged DTMUV.
Utility of multiplex reverse transcriptase-polymerase chain reaction for diagnosis and serotypic characterization of dengue and chikungunya viruses in clinical samples.
Baijayantimala Mishra, Mirnalini Sharma, Sujit Kumar Pujhari, Radha Kanta Ratho, Dvr Sai Gopal, Cvm Naresh Kumar, Gita Sarangi, Nirupama Chayani, Subhash C Varma
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India. email@example.com
The reemergence of chikungunya virus (CHIKV) has compounded the already existing dengue problem because of clinical similarities and common vector, demanding the need for a rapid and specific diagnosis. Thus, dengue chikungunya multiplex reverse transcriptase-polymerase chain reaction (DCmRT-PCR) was developed and validated for simultaneous detection of dengue and chikungunya viral infections and its utility in virus serotyping. Blood samples from 97 suspected dengue and chikungunya cases and 10 healthy controls were subjected to dengue and chikungunya conventional RT-PCR and DCmRT-PCR. Thirty-one of 97 samples were positive for dengue or chikungunya viral RNA by RT-PCR and DCmRT-PCR with 100% concordance. DCmRT-PCR products were cycle sequenced. Seven dengue virus strains were clustered within genotype III of DENV-3 and 4 within genotype III of DENV-1, whereas chikungunya sequences were clustered within the Central/East African genotype. DCmRT-PCR was found to be a potential rapid test for simultaneous detection of dengue and CHIKV in clinical samples along with dengue serotyping.
Detection of foot-and-mouth disease virus RNA in pharyngeal epithelium biopsy samples obtained from infected cattle: investigation of possible sites of virus replication and persistence.
National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected "carriers" of FMD-virus (FMDV) in ruminant species adds further complications to disease control. There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through measurements of the levels of FMDV RNA in the DSP as well as mandibular and retropharyngeal lymph nodes beyond 28 days after infection. Results indicated only low levels of FMDV RNA present in samples of pharyngeal epithelia during both early and persistent phases of infection with significantly higher levels of virus detected in pharyngeal excretions. It is concluded that the targeted area for sampling within the DSP does not harbour significant levels of virus replication during acute or persistent FMDV infection in cattle. Furthermore, the DSP and the mandibular and retropharyngeal lymph nodes cannot be concluded to be principal sites for persistence of FMDV.