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.

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.

Diversity and stability of Aleutian mink disease virus during bottleneck transitions resulting from eradication in domestic mink in Denmark.

Aleutian mink disease (plasmacytosis) virus (AMDV) in domestic mink (Neovison vison) has been subject to eradication in Denmark since 1976. In 2001, approximately 5% of Danish mink farms were still infected and all were located in the northern part of the peninsula of Jutland. In the present study a total of 274 Danish isolates of AMDV collected during the two seasons of 2004 and 2005 were characterized by partial sequencing of the coding region of the non-structural (NS) proteins. Older AMDV isolates from Denmark, available, were also included. The Danish isolates represent a very homogenous cluster compared with Swedish, Finnish and Dutch isolates and seem to represent a minor fraction of the genetic diversity previously found in Denmark. Stability of nucleotide deviations reveals that the purifying selection of bottlenecks imposed on the AMDV population in Denmark by the stamping out policy for more than 6 years exceeds the rate of mutation driven diversity. Among the isolates from farms in northern Jutland two distinct types could be identified and within each of them a number of sub-types which were all useful in tracking spread of infections. Infection at a farm the preceding season was a predisposing risk parameter for disease outbreak at a farm, and strain identity substantiates the suggestion that inadequate disinfection is involved in the recurrence of outbreaks. In cases of new introductions to farms it is indicated that contact including transport between farms played a most significant role.

Serotype specificity of antibodies against foot-and-mouth disease virus in cattle in selected districts in Uganda.

Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non-structural proteins and structural proteins. Three hundred and forty-nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non-structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified antibodies. High prevalences of antibodies against non-structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than against serotypes SAT 1, SAT 2 and SAT 3 in the sera investigated for serotype-specific antibodies. Only FMDV serotype O virus was isolated from one probang sample. This study shows that the majority of the FMD outbreaks in 2006 in the region studied were caused by FMDV serotype O; however, there was also evidence of antibodies to both SAT 1 and SAT 3 in one outbreak in a herd inside Queen Elizabeth national park area.

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.

Characterisation of foot-and-mouth disease virus strains circulating in Turkey during 1996-2004.

Two genotypes of foot-and-mouth disease virus serotype A were identified as the cause of disease outbreaks in Turkey during 1996-2004, while serotype O strains, identified during the same period, seem to represent an evolutionary continuum, and Asia1 strains were only rarely identified. The data presented are concordant with the conclusion that serotype A strains are repeatedly introduced to Turkey from the east and circulate only transiently in farming communities, while type O strains persist and re-emerge from endemic areas of Turkey. The co-circulation of strains belonging to two A genotypes for 6 years, as observed in the present study, is a remarkable difference compared to previous decades in which only one A genotype was transiently circulating, successively being replaced by others. This co-circulation was observed in spite of enforcement countrywide of biannual vaccination of more than 50% of the cattle during the same period. Mean r(1) values of 0.70 +/- 0.19 and 0.39 +/- 0.04 found for A96 and A99 isolates, respectively, compared to the A96 vaccine component reveal antigenic differences but also imply that the vaccine in use in Turkey should provide protection against both genotypes. It is suggested that further studies to reveal the nature of the difference in epidemiological dynamics of type A and type O strains might lead to an understanding of the measures required to control foot-and-mouth disease in islands of persistent circulation.

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.

Identification and partial characterization of Taastrup virus: a newly identified member species of the Mononegavirales.

We present a 8904-nt sequence of the central part of the RNA genome of a novel virus with a filovirus-like, nonidentical morphology named Taastrup virus (TV) detected in the leafhopper Psammotettix alienus. Sequence analysis identified five potential open reading frames (ORFs) and a complex pattern of homologies to various members of the Mononegavirales suggests a genome organization with the following order of genes: 3'-N-P-M-G-L-5'. Sequence analyses reveal an unusually large glycoprotein (G) containing both potential O-linked (14) and N-linked (9) glycosylation sites-a feature shared with the glycoproteins of Filoviridae and Pneumovirinae, and a nucleoprotein (N) with homology to the nucleoprotein of viral hemorrhagic septicemia virus (VHSV), a member of the Rhabdoviridae. Highly conserved domains were identified in the RNA-dependent RNA polymerase (L) between TV and other viruses within the order of Mononegavirales, and homology was found in particular with members of the Rhabdoviridae. The sequence similarities and the unique filovirus-like but nonidentical morphology unambiguously refer this newly identified virus to the order of Mononegavirales but to no family more than any, to other within the order.

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.

[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.

Search for Borna disease virus in Danish fibromyalgia patients.

OBJECTIVE: The purpose of this study was to look for Borna disease virus (BDV) in 18 patients with acute onset of fibromyalgia (FMS) following a "flu-like" episode. BDV is a neurotropic RNA virus affecting horses and sheep. Infections in animals have been reported to cause immune mediated disease characterized by abnormalities in behavior. A possible link between BDV and neuropsychiatric diseases in man has been described, and lately a connection to chronic fatigue syndrome (CFS) has been suggested. METHODS: A BDV-specific nested PCR (RT-PCR) was performed on serum and spinal fluid. RESULTS: The BDV genome was not detected in any of the FMS cases. CONCLUSION: Although BDV was not demonstrated in spinal fluid or serum from the tested patients with FMS, we believe that it is important to report our results, since FMS can exhibit many manifestations in common with CFS. Possible reasons for the discrepant findings are discussed.

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.

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.

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.

[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.

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.

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.