Real-time monitoring of Salmonella enterica in free-range geese.

Free-range geese were sampled longitudinally and Salmonella isolates characterized to reveal highly diverging colonization dynamics. One flock was intermittently colonized with one strain of Salmonella enterica serovar Enteritidis from 2 weeks of age, while in another, S. enterica serovar Mbandaka appeared after 9 weeks, without dissemination but with multiple serovars appearing at later stages.

Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.

A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

Molecular epidemiological study of Arctic rabies virus isolates from Greenland and comparison with isolates from throughout the Arctic and Baltic regions.

We report a molecular epidemiological study of rabies in Arctic countries by comparing a panel of novel Greenland isolates to a larger cohort of viral sequences from both Arctic and Baltic regions. Rabies virus isolates originating from wildlife (Arctic/red foxes, raccoon-dogs and reindeer), from domestic animals (dogs/cats) and from two human cases were investigated. The resulting 400 bp N-gene sequences were compared with isolates representing neighbouring Arctic or Baltic countries from North America, the former Soviet Union and Europe. Phylogenetic analysis demonstrated similarities between sequences from the Arctic and Arctic-like viruses, which were distinct from rabies isolates originating in the Baltic region of Europe, the Steppes in Russia and from North America. The Arctic-like group consist of isolates from India, Pakistan, southeast Siberia and Japan. The Arctic group was differentiated into two lineages, Arctic 1 and Arctic 2, with good bootstrap support. Arctic 1 is mainly comprised of Canadian isolates with a single fox isolate from Maine in the USA. Arctic 2 was further divided into sub-lineages: 2a/2b. Arctic 2a comprises isolates from the Arctic regions of Yakutia in northeast Siberia and Alaska. Arctic 2b isolates represent a biotype, which is dispersed throughout the Arctic region. The broad distribution of rabies in the Arctic regions including Greenland, Canada and Alaska provides evidence for the movement of rabies across borders.

A contribution to the systematization of bovine herpesvirus 1 based on genomic mapping by restriction fragment pattern analysis.

Fourteen isolates of bovine herpesvirus 1 (BHV-1) found representative of more than 100 isolates studied, were compared by restriction fragment pattern analyses and molecularly characterized. A number of evolutionary links between the variants originally associated with infectious bovine rhinotracheitis and the variants originally associated with infectious pustular vulvovaginitis were identified. These findings, as well as the lack of any correlation between genome type and clinical manifestation, confirm that there is no phylogenetic basis for a distinction between groups of strains associated with genital and respiratory disease. Two attenuated vaccine strains can be identified as deviating from field isolates.

Typing of European strains of parvovirus B19 by restriction endonuclease analyses and sequencing: identification of evolutionary lineages and evidence of recombination of markers from different lineages.

European isolates of parvovirus B19 were analyzed by restriction enzyme analysis of PCR products of the VP1/2 coding region and sequencing of the same amplified region, five cloned fragments from each PCR product. Two main groupings were found based on three perfectly linked point deviations. On the assumption that identical point deviations causing the various restriction patterns regardless of time and origin of virus isolation were unlikely to emerge independently in different evolutionary lineages, traits of evolutionary lineages were identified, suggesting a clonal population structure of global circulating B19 strains. However, combinations of markers from different evolutionary lineages were also found, particularly in a strain derived from an individual chronically infected with B19 for more than 7 years. As chronically infected individuals might be subject to superinfections due to contacts or possibly due to blood transfusions or the administration of gamma-globulin, it is suggested that coexistence of, and recombination between variants of B19 of different phylogenetic origin incidentally occur in such individuals.

Restriction pattern variability of respiratory syncytial virus during three consecutive epidemics in Denmark.

A PCR-based assay was used to distinguish between respiratory syncytial virus (RSV) group A and B in order to analyze their prevalence in Denmark in three consecutive epidemics during the winters of 1992/93, 1993/94 and 1994/95. A total of 96 RSV strains isolated from hospitalized children were examined, showing alternation of group prevalence. Furthermore, the genetic variability of the RSV isolates was illustrated by restriction enzyme analysis of PCR products originating from a part of the F and G proteins that has been reported to be highly variable. We found that, in general, different genome types predominated each year, some types being present in consecutive epidemics, indicating a contribution of strains circulating unattended between outbreak seasons, while others seemed to disappear or became undetectable, being replaced by emerging types. Some of the genome types found seemed related to strains isolated up to more than two decades ago in other parts of the world. This indicates that the temporal fluctuation in predominance of genome types presumably caused by selective pressure exerted by host immunity is due to the favoring of strains from a pool of globally circulating, genetically relatively stable genome types, rather than a molecular evolution in strains induced or directed by immunoselective pressure.

Sequence analysis of the hemagglutinin gene of measles virus isolates in Denmark 1997-1998: no evidence of persistent circulation of measles virus in Denmark.

The hemagglutinin-coding region of 17 virus samples from 12 measles cases in Denmark during 1997-1998 was analysed by partial nucleotide sequencing. The cases appeared as three sporadic cases and two epidemics, both with a limited time course and geographical distribution. The measles strains identified from the three sporadic cases and two epidemics could be allocated to five different previously well-defined sequence groups consistent with the assumption that cases of measles in Denmark are due to repeated introduction from abroad rather than persistent circulation of strains in the population.

The fluctuating pattern of various genome types of respiratory syncytial virus in Copenhagen and some other locations in Denmark.

A semi-nested RT-PCR method based on a region of the F and G glycoprotein genes was established, allowing the simultaneous detection and differentiation of group A and group B isolates of respiratory syncytial virus (RSV). The PCR products were subjected to digestion with restriction endonucleases to further differentiate the isolates. Using, in addition, previously reported studies the prevalence of various genome types in the Copenhagen region over a period of 6 years was established. Furthermore, the prevalence of genome types was determined in a distant region in Denmark during the winters of 1996/97 and 1997/98, and in yet another distant region during the winter of 1997/98. It was shown that the different regions in Denmark to a large extent share the same pool of genome types of RSV. Yet, while the fluctuating patterns of the two groups and various genome types were almost identical at different hospitals in the Copenhagen region, they varied between the different regions. This suggests that epidemics in local communities primarily rely on region-specific herd immunity parameters and emerge from strains endemically circulating in these local communities. Group B strains in Copenhagen showed an overall predominance, being predominant in three of the six epidemic seasons studied, and of almost equal predominance in one season.

Severity of respiratory syncytial virus disease related to type and genotype of virus and to cytokine values in nasopharyngeal secretions.

BACKGROUND: Investigations concerning the severity of respiratory syncytial virus (RSV) disease as related to (1) RSV type and genotype determined respectively by PCR and restriction enzyme analysis and (2) interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) values in samples of nasopharyngeal secretion (NPS) have not been previously reported. METHODS: We prospectively studied 105 RSV infections in the lower respiratory tract of infants and young children admitted to a pediatric department in Copenhagen during three winter seasons, 1993, 1994 and 1995. RSV strains were typed and genotyped, respectively, by PCR and nucleic acid restriction analysis and correlated to the severity of the disease. The ratio IL-6:TNF-alpha, determined from IL-6- and TNF-alpha values in samples of NPS, was related to the severity of the disease. Concentrations of IL-6 and of TNF-alpha were determined in serum samples taken during 5 weeks after the onset of illness. RESULTS: Type B infections produced more severe disease than did type A infections, as assessed on the length of the hospital stay, use of respiratory support and the presence of an infiltrate on a chest radiograph. This difference was age-related. It was observed in infants 0 to 5 months old, but not in older age groups. Type B genotype B1122 produced more severe disease than type A genotype A2311 in infants 0 to 11 months old. Increased serum concentrations of IL-6 and TNF-alpha were detected in samples taken 1 to 2 days after the onset of illness. Whereas TNF-alpha serum concentrations remained high, IL-6 serum concentrations decreased during the following 3 to 4 weeks. The IL-6:TNF-alpha ratio in samples of NPS was related to the severity of the disease. A high ratio was related to a low severity. CONCLUSIONS: The severity of disease in patients admitted with acute RSV infections can be correlated to the RSV type as determined by PCR, to the RSV genotype as determined by nucleic acid restriction analysis and to the ratio IL-6:TNF-alpha in NPS.

A rapid method for purification of herpesvirus DNA.

A rapid and reliable method for purification of herpesvirus DNA from cell cultures is described. The method is based on the isolation of virus particles and/or nucleocapsids by differential centrifugation and exploits the solubilizing and denaturing capabilities of cesium trifluoroacetate during isopycnic centrifugation, so that phenol/chloroform extractions can be omitted. The method was used for the purification of DNA from several members of the Alfaherpesvirinae subfamily.

Establishment of a genomic bank of bovine herpesvirus 1 using a novel positive selection plasmid vector.

A small positive selection cloning vector, designated pSiig1, suitable for the construction of genomic banks in E. coli is described and used for the establishment of a bank of the bovine herpesvirus 1 genome. Hybrid transformants are directly selected on agar plates containing ampicillin. The vector is based on the replicon of R1 and has a lambda PR promotor inserted upstream of the replication control genes. The vector has an uncontrolled (runaway) replication and is lethal to the host cell unless the PR promotor is brought under trans-acting control of the lambda cI repressor or runaway replication is blocked by an insertion between the PR promotor and the replicon. The vector contains a unique Bg/II site between PR and the replicon which is suitable for insertion of genomic DNA.

Persistent erythrovirus B19 urinary tract infection in an HIV-positive patient.

We report a urinary tract infection (UTI) with erythrovirus B19 in an HIV-1-positive homosexual man persisting for more than 7 months after the decline of viremia after a primary infection. During the course of the UTI, the patient complained of soreness in the kidney region and suffered from transient episodes of edema and hematuria. Proteinuria and elevated serum concentrations of creatinine further substantiated the hypothesis of a renal focus of a persistent erythrovirus B19 infection.

Analyses of restriction fragment patterns (RFPs) and pathogenicity in baby mice of equine herpesvirus 1 and 4 (EHV-1 and EHV-4) strains circulating in Danish horses.

Twenty-five strains of equine herpesvirus 1 (EHV-1) and one strain of equine herpesvirus 4 (EHV-4) isolated from material from various clinical cases in Denmark, together with reference EHV-1 and EHV-4 strains, were compared by restriction fragment pattern (RFP) analysis and inoculation of baby mice. The RFP analyses revealed that all EHV-1 strains belonged to genome type Ip. Four fetal isolates exhibited genomic characteristics that have been suggested as specific markers of the attenuated strain Rac H, widely used as a live vaccine. As the use of five vaccines against EHV-1 and EHV-4 has never been allowed in Denmark, it is assumed that Rac H derivatives have been acquired from visiting horses and thus are now circulating in the horse population. Baby mice inoculation revealed that four biotypes could be distinguished on the basis of pathogenicity. However, no strict correlation with pathogenicity in the natural host was seen.

The genomic diversity and stability of field strains of suid herpesvirus 1 (Aujeszky's disease virus).

The genomic diversity among isolates of suid herpesvirus 1 (SHV-1) collected in the same herd and among clones from the same isolate was studied by restriction fragment pattern (RFP) analysis using BamHI. Tentatively defining a field strain as a transmissible entity, it was concluded that strains of SHV-1 commonly comprise distinguishable genomic variants. Contrary to the hypothesis of genomic lability, it is suggested that the pool of variants is sufficiently stable to specifically characterize a strain. The impact of the results on epizootiological methods based on identification of field isolates is discussed in connection with Aujeszky's disease in Denmark.

Further evidence of long distance airborne transmission of Aujeszky's disease (pseudorabies) virus.

In spite of the eradication of Aujeszky's disease in Denmark a single outbreak was recorded in December 1988 and another severe epizootic took place during the winter and spring of 1989/90. The epizootic occurred in nearly the same areas as the preceding epizootic during the winter of 1987/88. Identification of the strains of virus involved eliminated the possibility that the latest epizootic was due to the persistence of virus in the pig population. Furthermore, as during the preceding epizootic, initial recordings of the new strains were found to coincide with periods with southerly winds. It was concluded from circumstantial evidence that the concurrent introductions of virus to several farms played a major role during the epizootic.

Evidence of long distance airborne transmission of Aujeszky's disease (pseudorabies) virus.

Aujeszky's disease has been the subject of an eradication campaign in Denmark since 1980. A detailed knowledge of the virus strains present in the country was provided by restriction fragment analyses of older clinical isolates, and of isolates from all the virologically confirmed outbreaks since 1985. The introduction of foreign strains into southern border areas was demonstrated during the winters of 1984/85, 1986/87 and 1987/88. An epizootic during the winter of 1987/88 was shown to correlate with an unusual predominance of southerly winds. Both conventional and specific pathogen free herds became infected. A herd level case-control analysis of the outbreaks during the winter of 1987/88 revealed that there was a positive correlation between the risk of infection and the size of the herd. The observations support the hypothesis of airborne transmission of the disease.

[Borna disease virus. An etiological agent in neuropsychiatric diseases?]. / Borna disease-virus. Et aetiologisk agens ved neuropsykiatriske lidelser?

Borna disease virus has long been recognized as a cause of sporadic cases and epidemics of meningoencephalomyelitis in horses and sheep in southern parts of Germany. however, sero-epidemiological surveillances indicate that Borna disease virus has a global distribution in horses, without the recognition of clinical manifestations associated with the infection, in other parts of the world. During the past five years evidence has been presented suggesting that humans also can become infected with this virus or a closely related virus. A significantly increased sero-prevalence is seen in patient populations with a variety of neuropsychiatric and behavioral disorders, suggesting that Borna disease virus or a closely related virus may play an etiological role in some of these diseases. A review of the literature is given.

[Cases of measles in Denmark are caused by reintroduction of virus from abroad]. / Maeslingetilfaelde i Danmark skyldes gentagne introduktioner af virus fra udlandet.

Measles vaccination was implemented in the child vaccination programme in Denmark in 1987 and produced a rapid decline in the incidence. Few cases were recorded annually until 1999. The measles virus isolated in Denmark during 1997-1998 was compared by partial sequencing of the haemagglutinin-coding region with Danish strains from the prevaccination era collected in 1965-1983, as well as with representatives of globally circulating strains of today. The dissimilarity of the prevaccination era strains identified in Denmark in 1997-1998 along with the similarity of these five strains with globally circulating strains at present, substantiate the conclusion that there is no persistent circulation of the measles virus in Denmark.

Low-cost monitoring of Campylobacter in poultry houses by air sampling and quantitative PCR.

The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 µm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 10(4) and 10(5) CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative real-time PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter.