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.

Natural and experimental infection of sheep with European bat lyssavirus type-1 of Danish bat origin.

In 1998 and 2002, European bat lyssavirus type-1 (EBLV-1) was demonstrated in brain tissue of five Danish sheep suffering from neurological disorders. Four of the five sheep also had encephalic listeriosis. The animals originated from four flocks on pastures within a limited area of western Jutland. In a serological investigation in two of the herds, from which three of the diseased animals originated, EBLV-1 neutralizing antibodies were detected in only one of 69 sheep. In follow-up surveys, 2110 sheep sera collected at Danish slaughterhouses during 2000 were all negative for EBLV-1-antibodies, and EBLV-1 was not demonstrated in 87 ruminants displaying neurological symptoms. To investigate the pathogenic effects of EBLV-1, four sheep were inoculated intralabially with either brain material from one of the naturally infected sheep or virus isolated from the same sheep. These animals developed EBLV-1 neutralizing antibodies at 5-9 weeks post-inoculation but did not exhibit neurological signs during a 33-week observation period. It was speculated that the immune response prevented viral dissemination to the brain, resulting in an abortive peripheral infection. It was concluded that EBLV-1 can infect sheep under natural conditions as an incidental event.

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.

Experiences from the Danish programme for eradication of bovine virus diarrhoea (BVD) 1994-1998 with special reference to legislation and causes of infection.

The main experiences from the Danish bovine virus diarrhoea (BVD) eradication programme over 5 years from 1994 to 1999 are presented. The last 3 years of the programme has been strongly supported by legislation. The most important regulations have been blood testing of live animals before movement to other herds, common pastures or exhibitions, and monitoring of all herds at regular intervals for the presence of the infection. Nevertheless, free herds have experienced infection, e.g., 204 dairy herds in 1998. Of herds found to be infected in the period from July 1997 through June 1998 after previously having been registered to be BVD-free, 67 herds were thoroughly investigated. Nineteen herds (28%) were found infected because of purchase of pregnant cows or heifers which delivered persistently infected (PI) calves, and 24 (36%) and two (3%) because of PI animals on neighbouring pastures or in neighbouring farm houses, respectively. In five herds (7%) pregnant heifers had become infected on one and the same common pasture, while in 17 herds (25%) no immediate cause of infection could be demonstrated. Yet, airborne spread from PI herds as a source of infection was suspected in some of these cases. It was furthermore concluded from investigations presented, that antibody-positive AI bulls were a remote but unlikely possibility. Free-living deer in Denmark had to be considered uninfected. Presence of PI-animals in sheep on infected farms has been seen and is paid attention to in individual cases. The results underline the need for legislation to be used in eradication programmes in areas with a high prevalence of infection and to be introduced right from the beginning in order to minimise the risk of infection for free herds.

Experimental in utero infection of pig foetuses with porcine parvovirus (PPV).

Pig foetuses of various gestational ages were exposed to experimental infection with porcine parvovirus (PPV) in utero. Inoculation of 40-, 50- and 60-day-old foetuses with PPV caused foetal death and mummification and spread of the infection to non-inoculated foetuses. Inoculation at 80 and 100 days gestation caused pathological lesions of various degrees whereas spread of infection occurred only sporadically. Serological examinations of foetuses of different ages suggest that immunocompetence for PPV develops before 70 days gestation. The present results strongly indicate that intrauterine spread of PPV is a route of transmission of this virus between pig foetuses.

Haemorrhagic disease of lagomorphs: evidence for a calicivirus.

Studies on the aetiological agents of rabbit haemorrhagic disease (RHD) and European brown hare syndrome show that the viruses responsible for these infections can be placed in the family Caliciviridae. Established members of this group are vesicular exanthema virus (prototype), San Miguel sea lion virus and feline calcivirus. The human hepatitis E virus and the Norwalk agent may soon be included. The RHD virus genome consists of a positive stranded RNA molecule composed of 7437 nucleotides. A major subgenomic RNA of 2.2 kb, colinear with the 3' end of the genomic RNA, can also be recovered from infected liver tissue, and both RNAs are enclosed within viral capsids formed by a single major protein of approximately 60 kDa. Electron microscopic examination of organ suspensions from diseased animals shows two types of particle; 35-40 nm complete virions have the regularly arranged cup-shaped depressions typical of calcivirus morphology, and 23-25 nm smooth particles resulting from degradation of the outer surface structures of the complete virions.

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.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at -5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%-67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus.

The ability of a pure virus infection to induce an acute phase protein response is of interest as viral infections are normally considered to be less efficient in inducing an acute phase protein response than bacterial infections. This was studied in a bovine model for infection with bovine respiratory syncytial virus (BRSV), analysing the induction of the two most dominant bovine acute phase proteins haptoglobin and serum amyloid A (SAA). Strong and reproducible acute phase responses were detected for both proteins, peaking at around 7-8 days after inoculation of BRSV, while no response was seen in mock-inoculated control animals. The serum concentrations reached for SAA and haptoglobin during the BRSV-induced acute phase response were generally the same or higher than previously reported for bacterial infections in calves. The magnitude and the duration of the haptoglobin response was found to correlate well with the severity of clinical signs (fever) and with the extent of lung consolidation while SAA responded most rapidly to infection.

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.

A retrospective evaluation of a Bovine Herpesvirus-1 (BHV-1) antibody ELISA on bulk-tank milk samples for classification of the BHV-1 status of Danish dairy herds.

Bulk-tank milk samples analysed in a Bovine Herpesvirus-1 (BHV-1) blocking ELISA are still in use in the Danish BHV-1 programme as a tool to classify dairy herds as BHV-1 infected or BHV-1 free herds. In this retrospective study, we used data from the Danish BHV-1 eradication campaign to evaluate performance characteristics of the BHV-1 blocking ELISA in 1039 BHV-1-seropositive and 502 repeatedly BHV-1-negative dairy herds using the results of blood testing of the individual animals as the true infection status. At a cut-off value of 30% blocking reaction, the herd-level relative sensitivity and relative specificity were 82 and 100%, respectively. The herd-level relative sensitivity depended on the within-herd prevalence of seropositive cows and the cut-off value in the assay, but not on the time interval (up to 90 days) between the collection of the bulk-tank milk sample and the individual serum samples. The BHV-1 blocking ELISA on bulk-tank milk could detect seropositive herds (few), with prevalence proportions as low as one seropositive cow out of 70 cows.

An experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus (BRSV) based on one combined exposure of calves.

Bovine respiratory syncytial virus (BRSV) has been recognised as an important pathogen in calf pneumonia for 30 years, but surprisingly few effective infection models for studies of the immune response and the pathogenesis in the natural host have been established. We present a reproducible experimental infection model for BRSV in 2-5-month-old, conventionally reared Jersey calves. Thirty-four colostrum-fed calves were inoculated once by aerosol and intratracheal injection with BRSV. Respiratory disease was recorded in 91% of the BRSV-inoculated calves, 72% had an accompanying rise in rectal temperature and 83% exhibited >5% consolidation of the lung tissue. The disease closely resembled natural outbreaks of BRSV-related pneumonia, and detection of BRSV in nasal secretions and lung tissues confirmed the primary role of BRSV. Nine mock-inoculated control calves failed to develop respiratory disease. This model is a valuable tool for the study of the pathogenesis of BRSV and for vaccine efficacy studies.

A novel subunit ISCOM vaccine against bovine virus diarrhoea virus.

The preparation and preliminary testing of a subunit ISCOM (immunostimulating complex) vaccine against bovine virus diarrhoea virus (BVDV) is described. Vaccination of calves with this vaccine yields high neutralising titres against a panel of Danish BVDV field isolates. The serological difference between virus isolates and vaccine strain selection is discussed.

A European comparative study of serological methods for the diagnosis of infectious bovine rhinotracheitis.

A comparison is made of serological diagnostic procedures used for infectious bovine rhinotracheitis in the European Community. A panel of 65 sera, including positive, doubtful, negative and diluted samples was dispatched to nine different European laboratories and tested by neutralisation, enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence and passive haemagglutination, according to the methods used in each laboratory. The results showed good agreement between the various tests for positive and negative sera. However, considerable variability between tests was noted in the "doubtful" category of sera. The highest test sensitivity, as judged by the detection limit for samples serially diluted in negative serum, was found with the blocking ELISAs. A need was identified for international standard reference sera to be made available.

Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.

European brown hare syndrome in free-ranging hares in Poland.

A study of European brown hare syndrome (EBHS) was conducted in Poland (Czempin). From April 1993 until February 1994, 100 blood and 78 spleen samples of European brown hares (Lepus europaeus) were tested for prevalence of EBHS and rabbit hemorrhagic disease (RHDV) antibodies and EBHS virus antigen with two enzyme-linked immunosorbent assay (ELISA) test kits. Thirty-eight of 100 serum samples were positive for EBHS, and six (7.6%) of 78 of the spleen materials were-antigen positive for EBHS virus. Three (3%) of the sera were positive against RHDV, whereas two of these also were seropositive for EBHSV. European brown hare syndrome virus seropositive hares were most frequently found from April to September. Based on negative staining electron microscopy investigations of liver and spleen homogenates of all antigen-positive hares, we observed caliciviruses in only one animal. For histopathological investigations organ specimens were available from 98 hares. Histopathological findings corresponded with the clinical picture of chronic EBHS. A pathohistological picture consistent with EBHS was found in 22 (22%) of investigated hares and corresponded in 50% of the animals which reacted positively in the EBHSV antigen-ELISA and in 29% of the animals which reacted positively in the EBHSV antibody-ELISA. These results from western Poland are the first that caliciviruses are present in European brown hare population in Eastern Europe and may be one of the causes for increased mortality in the Polish hare population over the past 10 years.

Control of bovine viral diarrhea virus infection without vaccines.

Successful control and eradication of BVDV infection presuppose sufficient knowledge of its epidemiology, particularly sources of infection and ways of transmission. Furthermore, it is crucial to have tests that can be trusted to give the true infection of individual animals and indicate the infection status of herds. PI animals are considered to be the main source of infection. In preliminary experiments in Denmark, it was found that eradication in herds could be based on the identification and removal of PI animals. Actual and possible means of herd-to-herd transmission of importance for infection control are reviewed. Special attention is paid to the possibility of airborne transmission, which must be anticipated in areas with high BVDV prevalence and a high cattle population density. BVDV control programs have been initiated only in the Scandinavian countries including Finland, where the occurrence of BVDV varies from a very low prevalence in Finland to a very high prevalence in Denmark. The BVDV programs in Finland, Norway, and Sweden are basically the same. The primary aim of each is the identification of the herds free from infection and prevention of introduction of the infection to these herds. A secondary aim is to reduce gradually the number of infected herds. In Denmark, which has a high prevalence of BVDV, the program is a combined control and eradication program, and different tests are used. The control programs in Scandinavian countries and the eradication program in Denmark are described together with the tests involved. With respect to control, special emphasis is given to herd tests applied to bulk tank milk or to specially selected blood samples to indicate the infection status of individual herds. The initial bulk tank milk testings were the main basis for the conclusions that in Finland, Norway, and Denmark approximately 1%, 9%, and 39% of the dairy herds, respectively, seemed to have PI animals. With respect to eradication, an ELISA developed in Denmark for demonstration of virus in blood has proved to be extremely reliable for identification of PI animals. The BVDV programs are generally voluntary, although in Norway, where BVD is a notifiable disease, restrictions have been placed on infected herds to prevent a further spread of the infection. The annual losses in Denmark from BVDV have been calculated to be approximately 100 million DKr (17 million dollars), whereas the total costs of the control and eradication program for a 3-year period including testing of trade animals are estimated to be approximately 160 million DKr (27 million dollars).

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.

Reintroduction of bovine herpes virus type 1 into Danish cattle herds during the period 1991-1995: a review of the investigations in the infected herds.

In Denmark a programme for the systematic eradication of bovine herpes virus 1 (BHV-1) was completed during the years 1984 to 1991, but outbreaks due to new introductions of BHV-1 were seen. Between January 1991 and May 1994, 22 herds became infected with BHV-1, all located closely to the German border. In 1995, 61 herds were detected BHV-1 antibody positive, but they were situated in many different parts of Denmark. In order to find the source of infection owners of infected herds were interviewed, and restriction fragment pattern analysis (RFP-analysis) was performed on virus isolates from the herds with clinical outbreaks. Isolates from clinical outbreaks up to 1995 were identified as a Cooper-like strain, while 2 of those in 1995 had characteristics of a "new" strain, which had never before been identified in Denmark or elsewhere in Europe. In the described situation different transmission routes for virus seemed possible. One being a sporadic introduction of virus due to accidental contact with infected cattle near the German border or maybe due to an airborne transmission of virus over longer distance. The other, presumably a result of import of an infected animal despite the national regulations. The latter, due to an extensive trade pattern, resulted in the introduction of infected cattle into 51 BHV-1 seronegative cattle herds.

Abortion and calf mortality in Danish cattle herds.

The aetiology of abortions and calf mortality in 65 Danish cattle herds consisting of both dairy and beef breeds during a 1-year period is described. All observed aborted foetuses, still-born calves, and calves dying before 6 months of age were necropsied, and relevant microbiological examinations were performed. A total of 240 calves and 66 abortions were submitted corresponding to a calf mortality rate of 7%. The abortion frequency could not be calculated. 43% of the calves died at day 0, while 22% were aborted, 15% died during the first week of life, 9% died from 1 to 4 weeks of age, and 11% died at the age of 1 to 6 months. The most common cause was neonatal pulmonic atelectasis (stillbirth) followed by foetal infections, pneumonia, and septicaemia.