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A pilot study was conducted to investigate the influence of electromagnetic fields in the short-wave range (3-30 MHz) radio transmitter signals on salivary melatonin concentration in dairy cattle. The hypothesis to be tested was whether EMF exposure would lower salivary melatonin concentrations, and whether removal of the EMF source would be followed by higher concentration levels. For this pilot study, a controlled intervention trial was designed. Two commercial dairy herds at two farms were compared, one located at a distance of 500 m (exposed), the other at a distance of 4,000 m (unexposed) from the transmitter. At each farm, five cows were monitored with respect to their salivary melatonin concentrations over a period of ten consecutive days. Saliva samples were collected at two-hour intervals during the dark phase of the night. As an additional intervention, the short-wave transmitter was switched off during three of the ten days (off phase). The samples were analyzed using a radioimmunoassay. The average nightly field strength readings were 21-fold greater on the exposed farm (1.59 mA/m) than on the control farm (0.076 mA/m). The mean values of the two initial nights did not show a statistically significant difference between exposed and unexposed cows. Therefore, a chronic melatonin reduction effect seemed unlikely. However, on the first night of re-exposure after the transmitter had been off for three days, the difference in salivary melatonin concentration between the two farms (3.89 pg/ml, CI: 2.04, 7.41) was statistically significant, indicating a two- to seven-fold increase of melatonin concentration. Thus, a delayed acute effect of EMF on melatonin concentration cannot completely be excluded. However, results should be interpreted with caution and further trials are required in order to confirm the results.

The neuroinvasiveness of California serogroup bunyaviruses is determined by the ability of the virus to replicate in striated muscle after peripheral inoculation of mice. Neuroinvasiveness was mapped to the medium (M) RNA segment of the virus, which encodes the viral glycoproteins, when reassortants were made between La Crosse/original virus, a neuroinvasive isolate, and Tahyna-181/57 virus, a nonneuroinvasive clone. We have tested the murine muscle cell line C2C12 as a surrogate for myotropism and have found that there is a slight, but reproducible difference in the replication of virus clones bearing the M RNA segment of La Crosse/original virus compared to clones bearing the M RNA segment of Tahyna-181/57 virus, as determined by viral titer, antigen expression, and plaque formation.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Due to the spread of bluetongue (BT) in Europe in the last decade, a sentinel surveillance programme was initiated for Switzerland in 2003, consisting of serological sampling of sentinel cattle tested for BT virus antibodies, as well as entomological trapping of Culicoides midges from June until October. The aim of this study was to create a 'suitability map' of Switzerland, indicating areas of potential disease occurrence based on the biological parameters of Obsoletus Complex habitat. Data on Culicoides catches from insect traps together with various environmental parameters were recorded and analysed. A multiple regression analysis was performed to determine correlation between the environmental conditions and vector abundance. Meteorological data were collected from 50 geo-referenced weather stations across Switzerland and maps of temperature, precipitation and altitude were created. A range of values of temperature, precipitation and altitude influencing vector biology were obtained from the literature. The final combined map highlighted areas in Switzerland which are most suitable for vector presence, hence implying a higher probability of disease occurrence given the presence of susceptible animals. The results confirmed the need for an early warning system for the surveillance of BT disease and its vectors in Switzerland.

Bovine anaplasmosis is a vector-borne disease that results in substantial economic losses in other parts of the world but so far not in northern Europe. In August 2002, a fatal disease outbreak was reported in a large dairy herd in the Swiss canton of Grisons. Diseased animals experienced fever, anorexia, agalactia, and depression. Anemia, ectoparasite infestation, and, occasionally, hemoglobinuria were observed. To determine the roles of vector-borne pathogens and to characterize the disease, blood samples were collected from all 286 animals: 50% of the cows were anemic. Upon microscopic examination of red blood cells, Anaplasma marginale inclusion bodies were found in 47% of the cows. The infection was confirmed serologically and by molecular methods. Interestingly, we also found evidence of infections with Anaplasma phagocytophilum , large Babesia and Theileria spp., and Mycoplasma wenyonii . The last two species had not previously been described in Switzerland. Anemia was significantly associated with the presence of the infectious agents detected, with the exception of A. phagocytophilum . Remarkably, concurrent infections with up to five infectious vector-borne agents were detected in 90% of the ill animals tested by PCR. We concluded that A. marginale was the major cause of the hemolytic anemia, while coinfections with other agents exacerbated the disease. This was the first severe disease outbreak associated with concurrent infections with vector-borne pathogens in alpine Switzerland; it was presumably curtailed by culling of the entire herd. It remains to be seen whether similar disease outbreaks will have to be anticipated in northern Europe in the future.

At the Swiss Federal Veterinary Office risk analyses are conducted according to international standards. A risk analysis contains the elements risk management, risk assessment and risk communication. A risk assessment is based on risk profile, hazard identification and a pathway model. All available information is gathered, documented and assessed and the risk estimated. The question. "What is the probability that unprocessed wild boar meat imported to Switzerland from the federal state Mecklenburg Western Pommerania is contaminated with classical swine fever virus?" was answered by a release assessment. The hazard identification recognized classical swine fever virus and attenuated live virus vaccine used for oral immunization as hazards. The probability of contamination was estimated to be small. The question: "What is the likelihood to introduce Aujeszky's disease to Switzerland and infect the indigenous pig population with the disease, by means of importing pork and meat products?" was answered by assessing the release, exposure and resulting consequences. The risk of an infection of the indigenous pig population was estimated to be very small, as 80% of the imported products derive from countries or zones free from Aujeszky's disease. Furthermore the majority of the imported products are processed. The strict implementation of the regulations governing feeding of food wastes to pigs reduces the probability of exposure. In all assessments the risk management decides on a strategy to deal with the risk, taking into consideration the results and recommendations derived from the risk assessment as well as other relevant factors.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) or prion disease of cattle first recognized in 1986 in the United Kingdom, where it produced a common source epidemic that peaked in January 1993 and has subsided markedly since that time. The epidemic began simultaneously at many geographic locations and was traced to contamination of meat and bone meal (MBM), a dietary supplement prepared from rendering of slaughterhouse offal. It appears that the epidemic was initiated by the presence of the agent of scrapie (a long-standing TSE of sheep) that was first transmitted to cattle, beginning in the early 1980s, when most rendering plants abandoned the use of organic solvents in the preparation of MBM. The epidemic was probably accelerated by the recycling of infected bovine tissues prior to the recognition of BSE. To terminate the epidemic, a prohibition on the feeding of ruminant-derived protein to ruminants was introduced in the United Kingdom in July 1988. The ruminant feed ban accounts for the decline of the epidemic after an interval of about 5 years, approximately equivalent to the average incubation period of BSE. Relatively few cases of BSE have occurred in cattle born after 1993, and it is predicted that the epidemic will terminate about the year 2000 based on an extrapolation of the present declining curve. A comparison of data from the United Kingdom with data from relatively low incidence countries, such as Switzerland, indicates that this epidemic has been mainly confined to the United Kingdom because of a unique concatenation of risk factors, including: 1) a high ratio of sheep to cattle; 2) a relatively high rate of endemic scrapie; 3) the heavy feeding of MBM to dairy cattle; and 4) changes in the rendering process used to prepare MBM. Recently, cases of a variant form of Creutzfeldt-Jakob disease (a TSE of humans) have been reported in the United Kingdom. These cases, at least 10 of which had onset in 1994-1995, are distinguished by their occurrence in subjects under age 40 years, by their clinical presentation, and by their neurohistopathologic picture. The appearance of this novel disease and its concentration in the United Kingdom have raised the question that it might represent the transmission of BSE to humans. However, the cases gave no history indicating an unusual exposure to live cattle, to the preparation of beef products, or of dietary exposure to bovine tissues, and it remains to be determined whether they are associated with BSE.

Summary: With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II - membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the ‘mycoides cluster’ or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the ‘mycoides cluster’.

Bovine spongiform encephalopathy (BSE), a subacute degenerative disease affecting the central nervous system of cattle, belongs to a group of related diseases known as the transmissible spongiform encephalopathies (TSEs). They are caused by poorly characterized scrapie-like agents (prions) that produce spongiform changes in the brain (1). Sheep scrapie is thought to be the origin of BSE, which was first diagnosed in the United Kingdom in 1986. The disease was confirmed a few years later in domestic cattle in France, Ireland, Portugal, and Switzerland (2), as well as in cattle exported from the United Kingdom to Denmark, Canada, the Falkland Islands, Germany, Italy, Oman, Portugal, and Ireland. Surveillance of BSE and/or scrapie is carried out in some countries. However, there is no test to detect the disease in live animals and the screening for the disease is rather difficult because it is based on the detection of clinically suspect cases (3). An additional approach to ascertain if BSE risk factors are likely to be present in a country is to conduct a qualitative or quantitative risk assessment. This chapter evaluates qualitatively some of the BSE risk factors in Europe.

La Crosse virus, a member of the California serogroup of bunyaviruses, is an important cause of pediatric encephalitis in the midwestern United States. Like all bunyaviruses, La Crosse virus contains two glycoproteins, G1 and G2, the larger of which, G1, is the target of neutralizing antibodies. To develop an understanding of the role of each of the glycoproteins in the generation of a protective immune response, we immunized 1-week-old mice with three different preparations: a vaccinia virus recombinant (VV.ORF) that expresses both G1 and G2, a vaccinia virus recombinant (VV.G1) that expresses G1 only, and a truncated soluble G1 (sG1) protein prepared in a baculovirus system. Whereas VV.ORF generated a protective response that was mostly directed against G1, VV.G1 was only partially effective at inducing a neutralizing response and at protecting mice from a potentially lethal challenge with La Crosse virus. Nevertheless, a single immunization with the sG1 preparation resulted in a robust immune response and protection against La Crosse virus. These results indicate that (i) the G1 protein by itself can induce an immune response sufficient for protection from a lethal challenge with La Crosse virus, (ii) a neutralizing humoral response correlates with protection, and (iii) the context in which G1 is presented affects its immunogenicity. The key step in the defense against central nervous system infection appeared to be interruption of a transient viremia that occurred just after La Crosse virus inoculation.

The California serogroup of viruses includes 14 viruses which take their name from California encephalitis virus, the prototype of the serogroup, which was isolated in California in the 1950s (Hammon and Reeves, 1952). This serogroup is part of the Bunyauirus genus within the family Bunyaviridae (Gonzalez-Scarano and Nathanson, 1990; Kolakofsky, 1991; Whitman and Shope, 1962). La Crosse virus, the most medically significant member of the California serogroup, was isolated in 1964 from a fatal case of encephalitis diagnosed in La Crosse, Wisconsin (Thompson et al., 1965). Although California encephalitis virus was initially reported in association with a few cases of encephalitis, it is not an important human pathogen. On the other hand, La Crosse virus is a common cause of encephalitis in the pediatric age group, with an average of about 75 reported cases of encephalitis each year (Kappus et al., 1983). In years when other arboviruses are not epidemic, La Crosse virus is the most important cause of arbovirus encephalitis in the United States. The virus is estimated to cause more than 100,000 inapparent infections annually which are concentrated in the midwest (Grimstad et al., 1984), which is the principal habitat of Aedes triseriatus, its major mosquito vector. Bunyavirions are roughly spherical particles about 100 nm in diameter consisting of a lipid envelope containing nucleocapsids representing the three different RNA species (L segment, 6.8 kb; M segment, 4.5 kb; and S segment, 0.9 kb) which comprise the negative-sense genome (Elliott, 1990; Gonzalez-Scarano and Nathanson, 1990). The viral envelope contains two glycoproteins, Gl and G2, and

The family Bunyaviridae is comprised of over 250 virus species divided into four genera: Bunyavirus, Phlebovirus, Hantavirus, and Nairovirus, and classified together because of conserved structural features (see below) (1, 2, 3, 4, 5). Most of the bunyaviridae are of no known medical or veterinary importance, and were isolated by routine inoculation of either mice or cell culture of arthropods obtained in the field (6). However, some viruses in several of the genera are important causes of human and animal diseases. Members of the California serogroup (genus Bunyavirus) and Rift Valley fever virus (genus Phlebovirus) can cause encephalitis, and are therefore of principal importance for this book. Recent reviews discuss the medical complications of infections with other Bunyaviridae (2, 7). Studies of the pathogenesis of central nervous disease have been mainly carried out with La Crosse and other members of the California serogroup, and our discussion will be largely confined to these. The members of the California serogroup are listed in Table 1.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.