Real-time PCR detection of Parvicapsula pseudobranchicola (Myxozoa: Myxosporea) in wild salmonids in Norway.

The myxozoan genus Parvicapsula contains 14 species infecting fish, some of which are known to cause severe disease in farmed and wild salmonids. Parvicapsula pseudobranchicola infections were first reported from seawater-reared Atlantic salmon, Salmo salar, in Norway in 2002 and have since then been an increasing problem. The present study describes a Taqman real-time PCR assay for specific detection of P. pseudobranchicola. The Taqman assay targets the 18S rRNA gene of P. pseudobranchicola and is able to detect as few as ten copies of the target sequence. Using the described assay, P. pseudobranchicola was detected in both farmed and wild salmonids, indicating that wild Atlantic salmon, sea trout, Salmo trutta, and Arctic char, Salvelinus alpinus, may be natural hosts of the parasite. Parvicapsula pseudobranchicola was found in samples from wild salmonids in the far south and the far north of Norway, displaying a wide geographic range of the parasite. Farmed salmonids showed P. pseudobranchicola infection levels many folds higher than that observed for wild sea trout, indicating that farmed Atlantic salmon are subjected to an elevated infection pressure compared with wild salmonids.

Epithelial distribution and replication of foot-and-mouth disease virus RNA in infected pigs.

Although the pathogenesis of foot-and-mouth disease (FMD) has been extensively investigated, relatively few studies have addressed the localization of FMD virus (FMDV) and in particular its replication in relation to the typical in-vivo sites of FMD lesions. In the present study, pigs were infected experimentally with FMDV serotype O UKG 34/2001 and tissue samples were collected from 1 to 4 days post-infection. Samples were stored at -70 degrees C and frozen sections were prepared for in-situ hybridization (ISH). A digoxigenin-labelled RNA probe complementary to a coding part of the RNA-dependent RNA polymerase (3D) genomic region was prepared. The FMDV positive strand RNA was prominent in the basal layers of the epithelium. A diffuse positive signal was also noted in the cytoplasm of cells of the stratum spinosum of lesional epithelium, but there was no signal in the stratum corneum. Detection of FMDV negative strand RNA was observed in basal cells above the basement membrane and along the dermal papillae. The basal cells therefore demonstrate the highest signal for detection of the FMDV positive and negative strand RNAs in both tongue and foot epithelium. These novel results suggest that the epithelial basal cells could be an early replication site of FMDV in vivo.

Dromedaries (Camelus dromedarius) are of low susceptibility to inoculation with foot-and-mouth disease virus serotype O.

Two sheep and five dromedaries were inoculated with a highdose of a cattle-passaged type O strain of foot-and-mouth disease virus (FMDV). The sheep developed typical FMD. The inoculated camels, which were placed in contact with five further dromedaries and four sheep, showed no visible signs of illness or vesicular lesions. However, one of them had a raised body temperature at 3 days post-inoculation (pi) and a viraemia from days 2 to 10; probang samples from this animal were negative for infectious virus, but a low level of FMDV RNA was detected in a sample taken on day 6 pi, five other samples taken from days 3 to 28 being negative. Examination of mouth swabs indicated a low level of FMDV RNA at days 1-5 pi in four of the five inoculated camels, but no infectious FMDV or FMDV RNA was detected in serum, probang or mouth swab samples from contact-exposed animals (camels and sheep). All the contact-exposed camels and sheep and two of the inoculated camels were serologically negative for FMD when tested up to day 28. In contrast, the camel with viraemia became serologically positive from day 14, and the other two inoculated camels (which had been exposed to FMDV in an earlier experiment) became serologically positive from day 10. The experiment suggested that dromedaries (1) are of low susceptibility to FMDV serotype O, (2) do not transmit infection, even by close contact, and (3) are unlikely to play a significant epidemiological role in FMD.

Temporal assessment of seroconversion in response to inactivated foot-and-mouth disease vaccine in Arabian oryx (Oryx leucoryx).

Ten male Arabian oryx (Oryx leucoryx) were vaccinated with a commercially available standard aqueous foot-and-mouth-disease vaccine containing aluminium hydroxide as an adjuvant, and their antibody titres against serotypes O and A were measured using solid-phase blocking elisa and the virus neutralisation test. Mean elisa antibody titres greater than 1.45 log(10) were recorded for serotype A, but low elisa titres were recorded for serotype 0; low titres were recorded by VNT for both serotypes.

Foot-and-Mouth Disease in Red Deer - Experimental Infection and Test Methods Performance.

The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Foot-and-mouth disease: host range and pathogenesis.

In this chapter the host range of foot-and-mouth disease (FMD) under natural and experimental conditions is reviewed. The routes and sites of infection, incubation periods and clinical and pathological findings are described and highlighted in relation to progress in understanding the pathogenesis of FMD.

Cytokine and Toll-like receptor mRNAs in the nasal-associated lymphoid tissues of cattle during foot-and-mouth disease virus infection.

The pharyngeal region is known to play an important role in foot-and-mouth disease virus (FMDV) infection in relation to acute disease and viral persistence. In this study, the local mucosal immune response in nasal-associated lymphoid tissue (NALT) of cattle infected with FMDV (strain O UKG 34/2001) was examined. Quantitative "real-time" RT-PCR assays were used to measure mRNA expression of cytokines (IFN-alpha, beta and gamma, IL-2, IL-1alpha and TNF-alpha) and Toll-like receptors (TLR)-3 and -4. NALTs from dorsal soft palate were collected from cattle at 7 days post-infection (dpi) and from carriers and non-carriers at 64 dpi. Expression of IFN-alpha mRNA was significantly greater in NALT during acute disease than in uninfected animals. Increased expression of IFN-gamma and IL-1alpha mRNA was also observed but was much lower than IFN-alpha expression. There was a slight increase in mRNA expression of TNF-alpha and IL-2. During persistence, TNF-alpha mRNA expression in carrier cattle was much higher than in non-carrier cattle. Expression of TLR-4 in NALT during the acute stage of infection was greater than in uninfected animals. Carrier and non-carrier cattle did not differ in respect of expression of TLR-3 and -4 mRNA in NALT.

Analysis of parvovirus infections using strand-specific hybridization probes.

The autonomous parvoviruses cause a broad spectrum of acute and chronic infections of animals and man. The discrimination of sites of viral replication from sites of viral sequestration is an important goal in elucidating the pathogenesis of these diseases. It is possible to employ strand-specific RNA hybridization probes in such analyses because a 'plus' sense probe will react with single stranded virion DNA and duplex replicative form DNA, but a 'minus' sense probe will react preferentially with obligate replicative intermediates (duplex replicative form DNA and mRNA). Strand-specific RNA hybridization probes were developed for the Aleutian mink disease parvovirus (ADV) and were used to study acute and chronic infections of mink. Such probes were capable of differentiating replicative intermediates (duplex replicative form DNA and mRNA) from single-stranded virion DNA in Southern blot analysis and in strand-specific in situ hybridization. ADV infection of seronegative newborn mink kits causes an acute, cytopathic infection of type II alveolar cells. Replication in these cells is highly permissive and is characterized by high levels of replicative intermediates and virion DNA. A fatal respiratory distress syndrome and hyaline membrane formation result from impaired surfactant production by the infected type II cells. On the other hand, ADV infection of adult mink is associated with a persistent infection and a disorder of the immune regulation. The target cells for viral replication in adult mink are confined to the lymphoid system and the bone marrow. Replication in these cells, which are probably lymphocytes, is restricted, and characterized by greatly reduced levels of replicative intermediates and virion DNA. It, therefore, seems that disease in the infected adult mink results from a restricted infection by ADV. Large amounts of virion DNA can also be demonstrated in locations where replication cannot be detected and apparently represents sequestration of virion particles by elements of the reticuloendothelial system. Thus, replication and sequestration can, in fact, be distinguished by the strand-specific in situ hybridization. These studies indicate that strand-specific in situ hybridization is a potentially valuable method for studying the pathogenesis of parvovirus infections.

Rocket line immunoelectrophoresis: an improved assay for simultaneous quantification of a mink parvovirus (Aleutian disease virus) antigen and antibody.

A rocket line immunoelectrophoretic assay (RLIE) was developed for the simultaneous quantification of viral antigens and antiviral antibodies of the important mink parvovirus, Aleutian disease virus (ADV). The sensitivity of the RLIE assay was found to be 5 log2 higher than that of the counter current immunoelectrophoresis which is the assay routinely used for diagnostic purposes.

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.

Validation of a foot-and-mouth disease antibody screening solid-phase competition ELISA (SPCE).

This paper describes the validation of a solid-phase competition enzyme-linked immunosorbent assay (SPCE) for the serological detection of antibody to serotype O foot-and-mouth disease (FMD) in sheep, cattle and pigs. The specificity of the SPCE was calculated from the results of testing known negative sera from sheep, cattle and pigs (n=3030, 1418 and 1495, respectively). The mean percentage inhibition (PI) for known negative sheep, cattle and pig sera were 19.3, 24.1 and 20.8%, respectively. The specificity of the SPCE at a cut-off point (COP) of 60 PI was 99.50% for sheep sera, 99.44% for cattle sera and 100% for pig sera. The analytical sensitivity of the SPCE was examined by testing sera from sheep, cattle and pigs. Based on the testing of serial bleeds from experimentally infected animals, seroconversion at the 60 PI COP occurred between 4 and 9 days post-infection or -exposure, similar to that observed using the virus neutralisation test (VNT) with a COP of 1/45. When applied to 267 sheep and 143 pig samples, that were obtained in Great Britain (GB) during the 2001 FMD UK outbreak, the SPCE identified more positive samples than did the VNT. Estimates of the accuracy, repeatability and reproducibility of the SPCE were verified during the large-scale serosurveillance necessitated by the 2001 outbreak. Results from field and experimental sera showed that when compared against the VNT, the sensitivity of the SPCE was less affected by the choice of virus strain used in the test. Using the O(1) UKG 2001 FMD virus in the VNT with samples representative of the uninfected GB sheep population, the test specificity was 100% at a COP of 1/45.

Serological and genetic characterisation of bovine respiratory syncytial virus (BRSV) indicates that Danish isolates belong to the intermediate subgroup: no evidence of a selective effect on the variability of G protein nucleotide sequence by prior cell culture adaption and passages in cell culture or calves.

Danish isolates of bovine respiratory syncytial virus (BRSV) were characterised by nucleotide sequencing of the G glycoprotein and by their reactivity with a panel of monoclonal antibodies (MAbs). Among the six Danish isolates, the overall sequence divergence ranged between 0 and 3% at the nucleotide level and between 0 and 5% at the amino acid level. Sequence divergences of 7-8%, 8-9% and 2-3% (nucleotide) and 9-11%, 12-16% and 4-6% (amino acid) were obtained in the comparison made between the group of Danish isolates and the previously sequenced 391-2USA, 127UK and 220-69Bel isolates, respectively. Phylogenetic analysis showed that the Danish isolates formed three lineages within a separate branch of the phylogenetic tree. Nevertheless, the Danish isolates were closely related to the 220-69Bel isolate, the prototype of the intermediate antigenic subgroup. The sequencing of the extracellular part of the G gene of additional 11 field BRSV viruses, processed directly from lung samples without prior adaption to cell culture growth, revealed sequence variabilities in the range obtained with the propagated virus. In addition, several passages in cell culture and in calves had no major impact on the nucleotide sequence of the G protein. These findings indicated that the previously established variabilities of the G protein of RS virus isolates were not attributable to mutations induced during the propagation of the virus. The reactivity of the Danish isolates with G protein-specific MAbs were similar to that of the 220-69Bel isolate. Furthermore, the sequence of the immunodominant region was completely conserved among the Danish isolates on one side and the 220-69Bel isolate on the other. When combined, these data strongly suggested that the Danish isolates belong to the intermediate subgroup.

Synovial fluid proteins in degenerative joint disease in dogs.

Concentrations of three immunoglobulins, albumin, ceruloplasmin, alpha-2 macroglobulin and pregnancy zone protein were estimated by immunoelectrophoresis in paired samples of synovial fluid and serum from 12 dogs with degenerative joint disease (DJD) and six normal dogs. The ratios of synovial fluid to serum concentrations (SF/S) of the four non-immunoglobulins showed an almost inverse linear relationship with their molecular weight in both groups. The SF/S were higher in the DJD synovial fluid than in normal synovial fluid. The difference increased with increasing molecular weight and was highly significant for the largest molecules, reflecting an increased permeability and inflammation in the synovial membrane of DJD joints. The SF/S ratios of the three immunoglobulins studied were compared to the diffusion curves of the four non-immunoglobulins. The SF/S ratios of IgM from dogs with DJD exceeded those calculated from the molecular weights. The present observations support the concept that DJD should be considered an inflammatory disease and suggest that immunologic processes may initiate and/or sustain the inflammation.

Increased pulmonary secretion of tumor necrosis factor-alpha in calves experimentally infected with bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory disease among calves in the Danish cattle industry. An experimental BRSV infection model was used to study the pathogenesis of the disease in calves. Broncho alveolar lung lavage (BAL) was performed on 28 Jersey calves, of which 23 were experimentally infected with BRSV and five were given a mock inoculum. The presence of the cytokine tumor necrosis factor alpha (TNF-alpha) in the BAL fluids was detected and quantified by a capture ELISA. TNF-alpha was detected in 21 of the infected animals. The amount of TNF-alpha in the BAL fluid of calves killed post inoculation day (PID) 2 and 4 was at the same very low level as in the uninfected control animals. Large amounts of TNF-alpha were detected on PID 6, maximum levels of TNF-alpha were reached on PID 7, and smaller amounts of TNF-alpha were seen on PID 8. The high levels of TNF-alpha appeared on the days where severe lung lesions and clinical signs were obvious and the amounts of BRSV-antigen were at their greatest. Although Pasteurellaceae were isolated from some of the BRSV-infected calves, calves treated with antibiotics before and through the whole period of the infection, as well as BRSV-infected calves free of bacteria reached the same level of TNF-alpha as animals from which bacteria were isolated from the lungs. It is concluded that significant quantities of TNF-alpha are produced in the lungs of the calves on PID 6-7 of BRSV infection. The involvement of TNF-alpha in the pathogenesis of, as well as the anti-viral immune response against, BRSV infection is discussed.

Determinants of early foot-and-mouth disease virus dynamics in pigs.

This paper provides a quantitative description of the early infectious process in pigs experimentally infected with foot-and-mouth disease virus (FMDV), obtained by dose-dependent, time course studies of viral load in serum. Pigs were inoculated by the intravenous or intradermal/subcutaneous route with FMDV and housed together in groups or individually. The effects of dose, inoculation route and exposure intensity on the replication of FMDV in vivo and the development of disease were studied. It was shown that the higher the dose, the shorter was the time to the start of active viraemia and to the onset of clinical signs. Exposure intensity and housing conditions influenced the viral dynamics of FMDV. Increasing the exposure intensity, by increasing the number of infected pigs housed together, had the effect of synchronizing the infection and reducing the variance in the start of active viraemia. Increasing the number of pigs housed together also increased the interaction between the pigs and the activity of individual pigs, which had the effect of shortening the time to the onset of clinical signs such as vesicle formation. Intradermal inoculation was more effective than intravenous inoculation for transmitting FMDV to pigs, resulting in shorter times to the start of active viraemia and in higher clinical scores.

Studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) can be spread by a variety of mechanisms and the rate of spread, the incubation period and the severity of disease depend on a multitude of parameters, including the strain of virus, the dose received, the route of introduction, the animal species and the husbandry conditions. More knowledge with regard to these parameters is urgently needed to improve resource-efficient disease control. This report describes detailed studies of FMDV load, excretion and transmission in pigs infected with FMDV O UKG 2001, O TAW 1997 and C Noville virus and in cattle infected with the O UKG 2001 virus to facilitate use of a "FMDV load framework" for the assessment of transmission risks. Virus replicated rapidly in pigs and cattle exposed by direct contact. The mean incubation period was around 3-4 days for cattle-to-cattle and 1-3 days for pig-to-pig transmission, depending on the intensity of contact. The results confirmed that a strong relation exists between dose and length of incubation period. Clinical disease was severe in pigs but relatively mild in inoculated cattle; contact infection of cattle appeared to increase the severity of lesions. FMDV RNA was recovered in nasal and mouth swabs from inoculated animals soon after they developed a viraemia and probably reflected the early production and excretion of virus. FMDV RNA in nasal and mouth swabs from contact animals could be detected several days before they showed other signs of infection, indicating the possibility of detecting exposed animals during the incubation period. FMDV RNA could also be detected in swab samples after the viraemic phase. This may have represented background environmental virus that had been trapped in the respiratory tract and mouth. Alternatively, it may have indicated a somewhat slower clearance or half-life of viral RNA or an extended low level of FMDV replication at these sites. The pattern of FMDV RNA concentrations in pigs was closely similar to that in cattle, but the amounts of FMDV RNA were higher.

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.

Use of automated real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor experimental swine vesicular disease virus infection in pigs.

Automated real-time RT-PCR was evaluated as a diagnostic tool for swine vesicular disease virus (SVDV) infection on a range of samples (vesicular epithelium, serum, nasal swabs, faeces) from four inoculated and three in-contact pigs over a period of 28 days. Traditional diagnostic procedures (virus isolation, and ELISAs for antigen and antibody) were used in parallel. Each inoculated pig developed a significant viraemia and clinical disease, and excreted virus, which was transmitted to the in-contact animals. The latter, however, developed only a short-lived, low-level viraemia and no clinical disease. The RT-PCR and virus isolation were generally comparable in detecting SVDV in the serum and nasal swabs from inoculated and in-contact pigs up to day 6 after infection; it was possible, however, to isolate virus for a longer period from the faeces of a few pigs. This suggested that further optimization of the template extraction method was required to counteract the effects of RT-PCR inhibitors in faeces. It was concluded that the automated real-time RT-PCR is a useful diagnostic method for SVD in clinically or subclinically affected pigs and contributed to the study of the pathogenesis of SVD in the pigs.

Clinical variation in foot and mouth disease: pigs.

In intensively reared pigs, the introduction of foot and mouth disease (FMD) results in severe clinical disease and vesicular lesions in adult and fattening animals, and high mortality in piglets. Vaccination of uninfected herds can assist FMD control and eradication programmes by reducing susceptibility of pigs older than 12 to 14 weeks and providing early protection to piglets through maternal antibody, but once FMD is established on a farm, vaccination alone will not prevent recurrent outbreaks of clinical disease.

Predicting the spread of foot and mouth disease by airborne virus.

Foot and mouth disease (FMD) can spread by a variety of mechanisms which, under certain climatic and epidemiological conditions, includes the windborne spread of disease. Recent advances in knowledge of the aerobiological features of FMD are described. The strain of virus and species of infected animal are major determinants of airborne virus emission. Pigs emit most virus, cattle and sheep lesser but similar amounts to each other. Peak excretion of airborne virus by sheep occurs before the clinical phase of disease, whereas with cattle and pigs, it coincides with the development of early clinical disease. The probability of aerogenous infection differs greatly between livestock species. Cattle are the most susceptible, followed by sheep, whereas pigs are very resistant. Computer-based simulation models have been developed to analyse and predict the risk of airborne spread of FMD and have been used successfully during outbreaks to support decision-making. Further research is required to refine and extend the models for operational use.