Åse Uttenthal

Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus.

Summary.  Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting the 3D gene of FMDV. The assay was validated for the efficacy to detect all known FMDV serotypes. The test method was linear over a range of at least 7 orders of magnitude and the detection limit was below the equivalent of 10 genomic copies. Analysing recent African probang samples the method was able to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon by melting point analysis for added specificity and at the same time allows the detection of mutations in the probe region. As such, the described new method is suitable for the robust real-time detection of index cases caused by any serotype of FMDV.

Replication and Clearance of Respiratory Syncytial Virus : Apoptosis Is an Important Pathway of Virus Clearance after Experimental Infection with Bovine Respiratory Syncytial Virus

Human respiratory syncytial virus is an important cause of severe respiratory disease in young children, the elderly, and in immunocompromised adults. Similarly, bovine respiratory syncytial virus (BRSV) is causing severe, sometimes fatal, respiratory disease in calves. Both viruses are pneumovirus and the infections with human respiratory syncytial virus and BRSV have similar clinical, pathological, and epidemiological characteristics. In this study we used experimental BRSV infection in calves as a model of respiratory syncytial virus infection to demonstrate important aspects of viral replication and clearance in a natural target animal. Replication of BRSV was demonstrated in the luminal part of the respiratory epithelial cells and replication in the upper respiratory tract preceded the replication in the lower respiratory tract. Virus excreted to the lumen of the respiratory tract was cleared by neutrophils whereas apoptosis was an important way of clearance of BRSV-infected epithelial cells. Neighboring cells, which probably were epithelial cells, phagocytized the BRSV-infected apoptotic cells. The number of both CD4+ and CD8+ T cells increased during the course of infection, but the T cells were not found between the epithelial cells of the bronchi up until apoptosis was no longer detected, thus in the bronchi there was no indication of direct contact-dependent T-cell-mediated cytotoxicity in the primary infection.

Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.

Sites of Replication of Bovine Respiratory Syncytial Virus in Naturally Infected Calves as Determined by In Situ Hybridization

Replication of bovine respiratory syncytial virus (BRSV) was studied in three naturally infected calves by in situ hybridization using strand-specific RNA probes. One of the calves was a 5-month-old Friesian, the other two calves were a 3-month-old and a 3-week-old Jersey. Two Jersey calves, 3 months and 3 weeks of age, served as controls. Replication of BRSV took place in the luminal lining of the respiratory tract. In one of the BRSV infected animals (calf No. 1), replication was especially seen in the bronchi, whereas in the two other animals (calf Nos. 2 and 3) replication of BRSV was demonstrated in the bronchiolar epithelial cells and in alveolar cells. Syncytia were often observed in the bronchiolar walls and in alveoli and such syncytia were always replicating BRSV. By immunohistochemistry it was possible to demonstrate BRSV antigen at the same location as replication of BRSV was detected. In tissue outside the respiratory tract neither BRSV antigen nor replication of BRSV could be demonstrated.

Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

Two recently developed vaccine--one based on synthetic peptide and one based on recombinant capsid protein--fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology ( > 98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based on inactivated virus. Surprisingly, this protection was obtained after only a single injection. Furthermore, the vaccinal dose of 150 micrograms of conjugated peptide or 3 micrograms of recombinant VP2 particles per animal, are sufficiently low to be cost-effective and applicable on a large scale.

Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.

Antibody dynamics in BRSV-infected Danish dairy herds as determined by isotype-specific immunoglobulins.

Abstract Using specific ELISAs, antibody levels of four different isotypes to bovine respiratory syncytial virus (BRSV) were determined in calves, following experimental BRSV infection. Most calves experienced an increase in the specific IgM and IgG1 titres about 6–10 days after infection with BRSV. The IgM titre was transient showing positive titres for only 5–10 days, while specific IgG1 was present for a longer time. IgA was detected concomitantly with IgM but at a lower level. Production of IgG2 anti-BRSV antibodies was detected from 3 weeks after infection. In two closed herds, repeated blood samplings were performed on young stock to analyse maternal immunity. The passively transferred antibodies were mainly of the IgG1 isotype and the half-life of IgG1 to BRSV was estimated to be 26.6 days. One of the herds had an outbreak of enzootic pneumonia, diagnosed to be caused by BRSV. Furthermore, another herd with acute BRSV was followed by weekly blood samples in six calves; in both herds IgM and IgG1 was detected shortly after the appearance of clinical signs. Serum samples from 50 Danish dairy herds (453 samples) were tested for immunoglobulins of the isotypes IgG1, IgG2 and IgM. The presence of antibodies to BRSV was widespread and more than 54% of the samples had BRSV antibodies of both the IgG1 and IgG2 isotypes indicating a high herd prevalence to BRSV. Test samples from two herds out of 50 were free from all isotypes to BRSV.

Comparing the epidemiological and economic effects of control strategies against classical swine fever in Denmark

In 2006, total Danish pork exports were valued at 3.8 billion euros, corresponding to approximately 5% of the total Danish exports, and an outbreak of a notifiable disease would have dramatic consequences for the agricultural sector in Denmark. Several outbreaks of classical swine fever (CSF) have occurred in Europe within the last decade, and different control strategies have been suggested. The objective of this study was to simulate the epidemiological and economic consequences of such control strategies in a CSF epidemic under Danish conditions with respect to herd demographics and geography and to investigate the effect of extra biosecurity measures on farms. We used InterSpread Plus to model the effect of nine different control strategies: the minimum measures required by the EU plus depopulation of contact herds (EUplus), extra depopulation of neighbouring herds, extra surveillance within the protection and surveillance zones, extra biosecurity in SPF herds-or in all herds, vaccination of all pigs in the 1 or 2 km zones using live vaccine as a protective measure (vaccination-to-kill), vaccination of all weaners and finishers in the 1 or 2 km zones using an E2 marker vaccine as a suppressive measure (vaccination-to-live). Each epidemic was simulated to start in four different index herds: production herds located in low, medium and high pig density areas, respectively; and a nucleus herd in an area of high pig density. For each control strategy and index case, we calculated the size and duration of the epidemic, the number of depopulated and/or vaccinated herds and animals, the control costs borne by the public and the pig industry, respectively, as well as the loss of exports associated with the epidemic. The simulations showed that the EUplus strategy is the most effective of the evaluated strategies with respect to limiting the size, duration and cost of the epidemic, regardless of the index case. However, regarding the number of slaughtered animals, the vaccination-to-live strategies appeared to be more effective. Epidemics become larger and last longer if the index case is a nucleus herd. This implies that biosecurity in nucleus herds is extremely important to avoid transmission of CSF to these herds. Simulations showed that a Danish CSF epidemic will be moderate in most cases and will include fewer than 10 cases and last less than 2 weeks on average. However, for some iterations, long-lasting and large epidemics were observed. Irrespective of the size and duration, an epidemic is expected to be very costly due to the export losses.

Characterisation of a pestivirus isolated from persistently infected mousedeer (Tragulus javanicus)

Summary. Serum samples from the male Mousedeer A and the mother, father and sister of A were tested for bovine virus diarrhoea viruses (BVDV) by isolation, and for BVDV antibodies by blocking ELISA and homologous neutralisation test. Further, RNA was extracted and tested by RT-PCR protocol analysing the 5′-untranslated region and the E2 gene of pestivirus. The RT-PCR products were subsequently sequenced. Mousedeer A was positive in virus isolation on three occasions (days 1, 19 and 40) and by RT-PCR. The sister and mother of Mousedeer A were also found virus positive by isolation and RT-PCR. Mousedeer A, its sister and its mother, all had an antibody neutralisation titer below 10. The father of A was virus negative but was positive in the blocking antibody ELISA and had a high neutralisation antibody titer. The repeated detection of BVDV in Mousedeer A, the high amount of virus in serum, the lack of antibodies and the virus positive family members documented that the mousedeer were persistently infected with a pestivirus. The father of A probably had an acute infection resulting in antibodies to pestivirus and viral clearance. Sequence analysis and phylogenetic analysis revealed that the mousedeer pestivirus was closely related to BVDV Type 1f. The existences of persistently infected animals in non-domestic species have great implications for BVDV eradication campaigns in cattle.