Soren Alexandersen

The Pathogenesis and Diagnosis of Foot-and-Mouth Disease

The pathogenesis of foot-and-mouth disease (FMD) is reviewed, taking account of knowledge gained from field and experimental studies and embracing investigations at the level of the virus, the cell, the organ, the whole animal and the herd or flock. The review also addresses the immune response and the carrier state in FMD. Progress made in understanding the pathogenesis of the disease is highlighted in relation to developments in diagnosis and methods of control.

Appearance of acute PRRS-like symptoms in sow herds after vaccination with a modified live PRRS vaccine

found in the dorsal root ganglia, the ventral horns of the spinal cord or the affected brainstem nuclei. As with other primary dysautonomias, the cause of the dysautonomia in the presented case was not determined. Despite extensive clinical, epizootiological and morphological investigations, mainly on feline and equine dysautonomia, the aetiology of any of these disorders is unknown. A neurotoxic agent is commonly suspected (Pollin and Griffiths 1992).

Aspects of the persistence of foot-and-mouth disease virus in animals—the carrier problem

Foot-and-mouth disease virus (FMDV) is a member of the Aphthovirus genus in the Picornaviridae family. Seven distinct serotypes, each including a wide range of variants, have been defined. FMD, affects wild and domesticated ruminants and pigs, is difficult to control and is the major constraint to international trade in livestock and animal products. After the acute stage of infection, FMDV may cause a prolonged, asymptomatic but persistent infection in ruminants. Also, vaccinated or naturally immune animals subsequently exposed to live virus may become persistently infected (the so-called carriers), a situation which can result in export embargoes if vaccination is included in a countrys control policy.

Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

Summary.Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 °C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV transcript were detected in twenty-two minutes. Amplification products were detected by visual inspection, agarose gel electrophoresis, or in real-time by the addition of a fluorescent dye. The specificity of the reaction was demonstrated by the absence of amplification of RNA from other viruses that cause vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan® assay, RT-LAMP appears to be sensitive, rapid, specific, and cost-effective, with the potential for field deployment and use by developing countries for FMDV surveillance.

The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative time-course study using TaqMan RT-PCR.

Foot-and-mouth disease (FMD) is a highly contagious, economically important virus disease of cloven-hoofed animals. The objective of the present study was to examine the early pathogenesis of FMD in pigs by a quantitative time-course study. Under experimental conditions, recipient pigs were infected by contact with donor pigs affected by FMD. Every 24 h from day 1 to day 4 after exposure, two recipient pigs were selected randomly, killed and necropsied. A range of tissues were analysed by a quantitative TaqMan RT-PCR method and by titration of FMD virus on primary bovine thyroid cells. The titres of virus determined by assay in cell culture and calculated from the quantitative TaqMan data correlated strongly (r>0.9), thereby establishing the validity of the TaqMan calculations. The data indicated that the replication of virus in the lungs contributes only in small part to airborne virus excretion. Sites in the pharynx, trachea and nasal mucosa are probably more important in that regard. The sites of earliest virus infection and possibly replication in recipient pigs appeared to be in the pharynx (soft palate, tonsil and floor of pharynx). The data indicated that FMD virus replication in pigs is rapid and that the majority of virus amplification occurs in the skin. A model for the progression of infection is proposed, indicating initial spread from the pharyngeal region, through regional lymph nodes and via the blood to epithelial cells, resulting in several cycles of virus amplification and spread.

Relative risks of the uncontrollable (airborne) spread of FMD by different species

SNOWDON, W. A. (1966) Growth of foot-and-mouth disease virus in monolayer cultures of calf thyroid cells. Nature 210, 1079 S0RENSEN, J. H., JENSEN, C. O., MIKKELSEN, T., MACKAY, D. K. J. & DONALDSON, A. I. (2001) Modelling the atmospheric dispersion of footand-mouth disease for emergency preparedness. Physics and Chemistry of the Earth B, 26, 93-97 SORENSEN, J. H., MACKAY, D. K. J., JENSEN, C. 0. & DONALDSON, A. I. (2000) An integrated model to predict the atmospheric spread of foot-andmouth disease virus. Epidemiology and Infection 124, 577-590 TINLINE, R. (1970) Lee wave hypothesis for the initial pattern of spread during the 1967-68 foot-and-mouth epizootic. Nature 227, 860-862

Development of a novel real-time RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues

Pigs are more difficult to immunise and more variable in their response to foot-and-mouth disease (FMD) than other livestock species. This has important consequences for FMD control during both prophylactic vaccination programmes in endemic situations and when emergency vaccination may be used as an adjunct to stamping out during outbreaks in countries normally free from the disease. The rapid and effective control of FMD in pigs is especially important in regions of high pig density since infected pigs have the potential to generate plumes of airborne virus and spread infection beyond the immediate control area. Increased knowledge of the kinetics of FMDV replication in pigs, especially in their respiratory tracts, could create opportunities for strategies to improve FMD vaccines for pigs. A fluorogenic TaqMan RT-PCR assay specific for the IRES (internal ribosome entry site) sequence of the O(1) Kaufbeuren/Lausanne strain of FMDV has been developed for this purpose. The assay had a sensitivity of 0.1 TCID(50)/ml, and a dynamic range of at least eight orders of magnitude. It was found that an established method of quantitation, relative to a housekeeping gene, exhibited two significant shortcomings when applied to a set of 16 anatomically highly diverse solid tissues: (i) tissue differences in housekeeping gene expression caused errors of up to 60-fold in estimated FMDV concentrations; and (ii) variability in total RNA yields caused unpredictable saturation of RT reactions, which in turn caused errors of up to 250-fold in the estimated FMDV concentration. A novel quantitation strategy, designated the C(t)(min) method, was developed to overcome these problems. The C(t)(min) method was based on the the RT-PCR examination of a dilution series of spectrophotometrically quantitated total RNA, spanning the optimum for the RT-PCR system used. The new method was influenced minimally by any tissue-specific RT-PCR inhibitors and was used to determine FMDV concentrations in tissues from four experimentally infected pigs. The results suggest that the lungs play a less important role in the replication of FMDV in pigs than was thought previously.

Genetic and Pathobiologic Characterization of Pandemic H1N1 2009 Influenza Viruses from a Naturally Infected Swine Herd

ABSTRACT Since its initial identification in Mexico and the United States, concerns have been raised that the novel H1N1 influenza virus might cause a pandemic of severity comparable to that of the 1918 pandemic. In late April 2009, viruses phylogenetically related to pandemic H1N1 influenza virus were isolated from an outbreak on a Canadian pig farm. This outbreak also had epidemiological links to a suspected human case. Experimental infections carried out in pigs using one of the swine isolates from this outbreak and the human isolate A/Mexico/InDRE4487/2009 showed differences in virus recovery from the lower respiratory tract. Virus was consistently isolated from the lungs of pigs infected with A/Mexico/InDRE4487/2009, while only one pig infected with A/swine/Alberta/OTH-33-8/2008 yielded live virus from the lung, despite comparable amounts of viral RNA and antigen in both groups of pigs. Clinical disease resembled other influenza virus infections in swine, albeit with somewhat prolonged virus antigen detection and delayed viral-RNA clearance from the lungs. There was also a noteworthy amount of genotypic variability among the viruses isolated from the pigs on the farm. This, along with the somewhat irregular pathobiological characteristics observed in experimentally infected animals, suggests that although the virus may be of swine origin, significant viral evolution may still be ongoing.

Reassortant Highly Pathogenic Influenza A H5N2 Virus Containing Gene Segments Related to Eurasian H5N8 in British Columbia, Canada, 2014

In late November 2014 higher than normal death losses in a meat turkey and chicken broiler breeder farm in the Fraser Valley of British Columbia initiated a diagnostic investigation that led to the discovery of a novel reassortant highly pathogenic avian influenza (HPAI) H5N2 virus. This virus, composed of 5 gene segments (PB2, PA, HA, M and NS) related to Eurasian HPAI H5N8 and the remaining gene segments (PB1, NP and NA) related to North American lineage waterfowl viruses, represents the first HPAI outbreak in North American poultry due to a virus with Eurasian lineage genes. Since its first appearance in Korea in January 2014, HPAI H5N8 spread to Western Europe in November 2014. These European outbreaks happened to temporally coincide with migratory waterfowl movements. The fact that the British Columbia outbreaks also occurred at a time associated with increased migratory waterfowl activity along with reports by the USA of a wholly Eurasian H5N8 virus detected in wild birds in Washington State, strongly suggest that migratory waterfowl were responsible for bringing Eurasian H5N8 to North America where it subsequently reassorted with indigenous viruses.

Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada.

Summary In January 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (PED) in the USA, the first case of PED was confirmed in a swine herd in south‐western Ontario. A follow‐up epidemiological investigation carried out on the initial and 10 subsequent Ontario PED cases pointed to feed as a common risk factor. As a result, several lots of feed and spray‐dried porcine plasma (SDPP) used as a feed supplement were tested for the presence of PEDV genome by real‐time RT‐PCR assay. Several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of PEDV into Canada. These findings led us to conduct a bioassay experiment in which three PEDV‐positive SDPP samples (from a single lot) and two PEDV‐positive feed samples supplemented with this SDPP were used to orally inoculate 3‐week‐old piglets. Although the feed‐inoculated piglets did not show any significant excretion of PEDV, the SDPP‐inoculated piglets shed PEDV at a relatively high level for ≥9 days. Despite the fact that the tested PEDV genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role.