Redefining the "carrier" state for foot-and-mouth disease from the dynamics of virus persistence in endemically affected cattle populations.

The foot-and-mouth disease virus (FMDV) "carrier" state was defined by van Bekkum in 1959. It was based on the recovery of infectious virus 28 days or more post infection and has been a useful construct for experimental studies. Using historic data from 1,107 cattle, collected as part of a population based study of endemic FMD in 2000, we developed a mixed effects logistic regression model to predict the probability of recovering viable FMDV by probang and culture, conditional on the animal's age and time since last reported outbreak.

Identification of radically different variants of porcine reproductive and respiratory syndrome virus in Eastern Europe: towards a common ancestor for European and American viruses.

We determined 22 partial porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 sequences, representing pathogenic field strains mainly from Poland and Lithuania, and two currently available European-type live PRRSV vaccines. Also, the complete ORF7 of two Lithuanian and two Polish strains was sequenced. We found that Polish, and in particular Lithuanian, PRRSV sequences were exceptionally different from the European prototype, the Lelystad virus, and in addition showed a very high national diversity.

A longitudinal study of cell-mediated immunity in pigs infected with porcine parvovirus.

Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV infection remains to be elucidated, and may be relevant to the pathogenesis of novel diseases such as postweaning multisystemic wasting syndrome (PMWS), which may be triggered by coinfection with PPV and porcine circovirus type 2 (PCV2).

Porcine B-cells recognize epitopes that are conserved between the structural proteins of American- and European-type porcine reproductive and respiratory syndrome virus.

By selecting phage display libraries with immune sera from experimentally infected pigs, porcine B-cell epitopes in the open reading frame (ORF) 2, 3, 5 and 6 proteins of European-type porcine reproductive and respiratory syndrome virus (PRRSV) were identified. The sequences of all the epitopes were well conserved in European-type PRRSV and even between European- and American-type PRRSV. Accordingly, sera from pigs infected with American-type PRRSV cross-reacted with the European-type epitopes.

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I).

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli.

Experimental inoculation of late term pregnant sows with a field isolate of porcine reproductive and respiratory syndrome vaccine-derived virus.

The use of a live attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in piglets has been associated with reproductive disorders in non-vaccinated sows. Vaccine-derived virus (VDV) has been isolated from foetuses, stillborn pigs, and dead piglets, indicating that the live vaccine spread from vaccinated piglets to non-vaccinated sows, and that the virus might be implicated in the severe reproductive problems observed. In the present study, one such VDV isolate was used to experimentally infect pregnant sows in the last trimester.

High frequency RNA recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity.

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) exist, a North American type and a European type. The co-existence of both types in some countries, such as Denmark, Slovakia and Canada, creates a risk of inter-type recombination. To evaluate this risk, cell cultures were co-infected with either a North American and a European type of PRRSV or two diverse types of European isolate.

Classical swine fever (CSF) marker vaccine. Trial I. Challenge studies in weaner pigs.

Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany.

Semen from boars infected with porcine reproductive and respiratory syndrome virus (PRRSV) contains antibodies against structural as well as nonstructural viral proteins.

The seminal excretion of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) was examined in a group of five boars experimentally infected by the nasopharyngeal route. By using phage-displayed peptide epitopes from the PRRSV replicase and envelope glycoproteins as ELISA antigen, we were able to separately and specifically assay antibody responses against structural and nonstructural viral proteins. Antibodies against structural as well as nonstructural viral proteins were consistently found in the semen of all boars, beginning from 1-4 weeks postinfection.

Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations.

A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates was sequenced and compared with the parental strain of the vaccine virus (VR2332). This revealed five mutations that had occurred independently in all three vaccine-derived field isolates, indicating strong parallel selective pressure on these positions in the vaccine virus when used in swine herds.

Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes.

We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11 to 53 amino acids in length. In the replicase polyprotein, a total of eight ES were identified, six of which localized to the Nsp2 replicase polyprotein processing end product.

Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotides.

Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species.

Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis.

A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism analysis.

Preparation and characterisation of quillaja saponin with less heterogeneity than Quil-A.

Immunisation against pathogens remains one of the most effective ways of preventing or reducing losses due to infectious diseases in animal husbandry. When inactivated vaccines are used, adjuvants are most often required to obtain satisfactory immune responses. One such type of adjuvant is saponin derived from the bark of Quillaja saponaria Molina, a tree of the rose family.

Examination of the selective pressures on a live PRRS vaccine virus.

We determined the ORF5 and 7 sequences of 20 pathogenic revertants of a live PRRSV vaccine. The sequence analysis confirmed all 20 isolates to be of vaccine origin. Having established that clonal introduction of American (vaccine) PRRS virus had occurred in Denmark, we could perform analysis of the selective pressure this attenuated virus had experienced during reversion.

Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies.

Many methods have been devised for double immunocytochemical staining. We now describe that moderate microwaving does not elute antibodies, but prevents their reactions with subsequently applied reagents. Thus, microwaving performed in between the first and second staining cycles permits double indirect immunofluorescence staining with antibodies raised in the same species.

Emergence of porcine reproductive and respiratory syndrome virus deletion mutants: correlation with the porcine antibody response to a hypervariable site in the ORF 3 structural glycoprotein.

By using porcine immune sera to select a library of phage-displayed random peptides, we identified an antigenic sequence (RKASLSTS) in the C-terminus of the ORF 3 structural glycoprotein of European-type porcine reproductive and respiratory syndrome virus (PRRSV). Through the use of overlapping reading frames, the same PRRSV genetic locus codes for the ORF 3 "RKASLSTS" sequence, and a previously described ORF 4 epitope (Meulenberg, J. J.

Designing serological surveillance programmes to document freedom from disease with special reference to exotic viral diseases of pigs in Denmark.

Surveillance programmes based on laboratory screening tests are increasingly used to document freedom from disease in order to facilitate trade. The following aspects must be considered when designing such programmes: diseases to be selected; epidemiology of the diseases; unit of analysis (animal or herd); target age group (or target farm type); test characteristics and sample size. Issues related to these aspects are discussed and illustrated using the example of serological surveillance for exotic viral diseases in the pig population of Denmark.

Experimental corticosteroid induction of Pneumocystis carinii pneumonia in piglets.

Animal models of Pneumocystis carinii (Pc) pneumonia (PCP) play a central role in research on the Pc microorganism itself and the disease, especially the pathogenesis and the host defence. The classic rat model with corticosteroid-induced reactivation of a latent infection has been most widely used. In our search for alternative non-rodent models, six 31/2-week-old piglets were injected intramuscularly with methylprednisolone acetate, at 18 mg/kg body weight, once a week for 6 weeks.

Heterologous challenge with porcine reproductive and respiratory syndrome (PRRS) vaccine virus: no evidence of reactivation of previous European-type PRRS virus infection.

In Denmark, a porcine reproductive and respiratory syndrome virus (PRRSV) control programme, comprising vaccination of seropositive herds with a live American type PRRSV vaccine, was started in 1996. In several of these herds, spread of vaccine virus from vaccinated 3-18 week old pigs to non-vaccinated sows was demonstrated by the isolation of vaccine virus from fetuses and stillborn piglets. Surprisingly, sows infected with the American type vaccine strain consistently exhibited significantly stronger serological responses towards European type PRRSV than American type PRRSV.

Determination of 5'-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs.

We determined the untranslated 5'-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5'-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication.

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on alveolar lung macrophage survival and function.

Porcine reproductive and respiratory syndrome virus (PRRSV) recently emerged as an important cause of reproductive disorders and pneumonia in domestic pigs throughout the world. Acute cytocidal replication of PRRSV in alveolar lung macrophages causes the acute pneumonia; however, it remains largely unresolved whether there may also be a predisposition to longer-term local immunodeficiency in the PRRSV-convalescent lung. We applied various flow cytometric techniques to study the interplay between PRRSV replication and macrophage viability/function in pure cultures of porcine alveolar lung macrophages.

Production of a highly immunogenic subunit ISCOM vaccine against Bovine Viral Diarrhea Virus.

Bovine Viral Diarrhea Virus (BVDV) is a major pathogen of cattle in most countries. The main reservoir of virus in herds are BVDV persistently infected animals, which arise as a result of infection of the bovine fetus early in gestation. The spread of virus to the unborn fetus may be prevented by vaccination of the dam.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes.

Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV.

Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds.

Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds.