Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus.

Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its economic importance, programs to eradicate bovine viral diarrhea are a high priority in the cattle industry. By contrast, Border disease is not a target of eradication, although the Border disease virus is known to be capable of also infecting cattle. In this work, we compared single dose experimental inoculation of calves with Border disease virus with co-mingling of calves with sheep persistently infected with this virus. As indicated by seroconversion, infection was achieved only in one out of seven calves with a dose of Border disease virus that was previously shown to be successful in calves inoculated with BVD virus. By contrast, all calves kept together with persistently infected sheep readily became infected with Border disease virus. The ease of viral transmission from sheep to cattle and the antigenic similarity of bovine and ovine pestiviruses may become a problem for demonstrating freedom of BVD by serology in the cattle population.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland.

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

Le but du présent travail était de savoir si, dans des exploitations détenant en parallèle des bovins et des moutons, on trouve des moutons infectés de façon persistante par la Border Disease (BD) et, dans ce cas, si les bovins de ces exploitations présentaient des anticorps contre la BVD. En outre on cherchait à connaître la séroprévalence des moutons quant aux anticorps BDV et BVDV. Les recherches ont été menées dans 76 exploitations détenant des moutons et des bovins. 2'384 échantillons sanguins de moutons ont été testés par PCR quantitative en temps réel quant aux pestivirus et 2'291 par ELISA quant aux anticorps contre les pestivirus. 27 autres échantillons, positifs à l'ELISA et provenant de 10 exploitations, ont été soumis à un test de séroneutralisation, afin de savoir si les anticorps étaient dirigés contre le BDV ou le BVDV. Chez les moutons dont le titre contre le BDV était au moins quatre fois plus élevé que celui contre le BVDV, on a considéré qu'il s'agissait d'une infection avec le BDV. Le titre BVDV a été évalué de la même manière. Tous les moutons testés quant aux pestivirus étaient virologiquement négatifs. Dans la recherche par ELISA, 310 échantillons étaient positifs, 119 douteux et 1'862 négatifs. La séroprévalence des exploitations variait entre 0.0 et 73.9 %. Lors de l'analyse par séroneutralisation des 27 échantillons positifs à l'ELISA, 6 échantillons provenant de 3 exploitations présentaient un titre BDV plus de quatre fois plus élevé que celui de BVDV. 14 échantillons provenant de 6 exploitations montraient des titres BVDV plus de quatre fois plus élevés que ceux de BDV. Sur la base de ces résultats, on doit admettre dans 3 exploitations une infection des moutons par BDV et dans 6 une infection par BVDV.

Infection of cattle with Border disease virus by sheep on communal alpine pastures.

The purpose of this study was to investigate whether sheep grazing communal alpine pastures with cattle can transmit Border disease virus (BDV) to cattle. A total of 1170 sheep and 923 cattle were tested for BDV using RT-PCR (sheep) and for pestivirus antibodies using an ELISA (cattle), respectively, before being moved to one of 4 pastures (A, B, C and D). Eight sheep from pasture C were viraemic. 396 of 923 cattle examined before the pasture season were seronegative. The latter were re-examined after the pasture season and 99 were seropositive or indeterminate. Antibody specificity was determined in 25 of these using a serum neutralization test (SNT). BDV infection was confirmed in 10 cattle and was considered likely in 8 others. BVDV infection was confirmed in 4 cattle and considered likely in 3 after pasturing. The study has shown that the transmission of BDV from sheep to cattle is possible on communal alpine pastures.

Mucosal lesions in a sheep infected with the Border Disease Virus (BDV).

A 28-week-old sheep was presented at the animal hospital because of chronic emaciation, anemia and slight diarrhea. Due to poor general condition and bad prognosis the animal was euthanized and submitted for postmortem investigation. Multiple erosions and ulcerations were found in the dorsal region of the tongue, the pharynx, the hard palate, in the esophagus and the ruminal pillars. Histologically, these lesions consisted of necrosuppurative inflammation. The animal was tested positive for pestivirus antigen both by immunohistochemical and by virological examination (cell culture, antigen capture ELISA and RT-PCR). A non-cytopathic Border Disease Virus was identified, and sequencing revealed a virus belonging to the BDV-3 cluster. Based on the macroscopical, histological, immunohistological and virological results this case was diagnosed as Border Disease with mucosal lesions. This is the first report of such a case in Switzerland.

Seroprevalence and characterization of pestivirus infections in small ruminants and new world camelids in Switzerland.

The seroprevalence of pestivirus infections in small ruminants and new world camelids in Switzerland was determined. In 5'059 sera of sheep from 382 herds, 503 sera of goats from 54 herds and 109 sera of alpacas and lamas from 53 herds, population prevalences of 16.1% (sheep), 25.4% (goats) and 4.6% (new world camelids), respectively, were found. In order to determine the source of infection, the serological reactions were further characterized by cross-neutralization against two pestiviruses representing the genotypes BVDV (Bovine Virus Diarrhea Virus)-1 and BDV (Border Disease Virus)-1. Based on the ratio of respective antibody titres, 56.1% of the infections in sheep were induced by a BDV-1, 12.9% by a BVDV-1 and 31.0% by an unresolved pestivirus. In goats, the corresponding proportions were 23.4%, 10.2% and 66.4%, respectively. In Alpacas and Lamas, the source of infection of 1 animal was BDV-1 and that of 4 seropositive animals remained unresolved. In view of the phylogenetic relationship between pestiviruses, the unresolved source of infection is most probably attributable to other pestivirus genotypes circulating in small ruminants and new world camelids. Due to the predominance of pestiviral genotypes other than BVDV-1, the risk of transmission of BVDV from persistently infected small ruminants and new world camelids to cattle appears to be moderate, apart from close direct contact in mixed animal husbandry, communal pasturing and grazing in the Alps.

[Experiences with a BVD-free transhumance in the summer of 2006]. / Erfahrungen mit einer BVD-freien Alpung im Sommer 2006.

The aim of this paper was to examine the effect of eliminating persistently infected (PI) animals on BVDV infection during transhumance and to identify possible weak points in the prevention of new infection. An initial blood sample (A) was taken from all the animals until one week before the date of trans-humance (n = 190) and examined for virus by means of real-time RT-PCR or antigen-ELISA and for antibodies by means of ELISA. One PI animal was identified and eliminated. On the day of transhumance (B), serology was performed of the blood samples of all animals that had had a negative or unknown antibody status (n = 93) when blood sample A had been examined. At the end of the transhumance season (C) those animals that had tested seronegative in sample B were re-examined for antibodies (n = 65). The case incidence per animal year amounted to 37.1% up to sample A, 41.8% between sample A and sample B (4 seroconversions). Four cases of seroconversion were diagnosed during the transhumance season, which equalled a case incidence of 17.8% per animal year. A season of transhumance free of PI animals failed to completely prevent BVDV infection, but the new infection rate was significantly diminished. The most possible explanation for new infections are abortions of PI-animals.

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies.

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

[Heifer breeding farms as a source for spreading the BVD virus?]. / Aufzuchtbetriebe als Verbreitungsquelle des BVD-Virus?

It is well known that, in Switzerland, communal grazing of livestock on alpine pastures plays an important role in the spread of BVD virus. Analogously, we might expect that the communal raising on farms specialising in raising heifers of animals born on different farms would also favour the spread of BVDV. This study investigated whether a persistently infected (PI) breeding heifer kept on this type of farm over a period of 26 months would put the other animals at risk of being infected. The PI-animal was in contact with 75 heifers (here defined as contact animals) on this farm. Thirty-two of the contact animals that were probably pregnant (animals at risk of giving birth to a PI-calf) were moved to 8 different breeding farms (here defined as farms at risk). On these 8 farms, 246 calves were found to be at risk of being infected with BVDV. We examined 78 calves and investigated whether the move of the pregnant animals from their original farm had permitted the virus to spread to these 8 other farms. The contact animals had a seroprevalence of 92% and the animals at risk a seroprevalence of 100%. Only one PI-animal was found on the farms at risk. This BVD infection, however, occurred independently of the PI-breeding animal. Seropositive calves were found only on 2 farms. This study did not provide any proof for a spread of BVDV with the PI-breeding animal as a source; likewise, no persistent infection was proven to exist on the farms at risk. This result is likely to be representative for the endemic situation of BVD in Switzerland. Thus, PI-animals present on heifer raising farms infect calves well before servicing. Hence, no new PI-animals are generated, and the infection becomes self-limiting. When we reconstructed the animal movements between the farms and determined the animals to be examined with the aid of the Swiss national animal traffic database (TVD) we found the data of 37% of the heifers to be incomplete and failed to successfully establish the whereabouts of 3 animals.

[How the bovine viral diarrhea virus outwits the immune system]. / Wie das Bovine Virusdiarröe-Virus das Immunsystem überlistet.

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.

Genetic heterogeneity of pestiviruses of ruminants in Switzerland.

We have genetically analyzed ruminant pestiviruses. All >150 bovine viral diarrhea (BVD) viruses isolated from cattle in Switzerland belonged to genotype 1, with subgenogroups e, h, k and b found in decreasing frequency. To date, representatives of subgenogroup k have been detected in Switzerland only. Despite serological evidence of Border disease in sheep, only few Border disease viruses have been isolated, all of which belong to the novel group 3. Serological evidence suggested that pestivirus infections may occur also in wild ruminants in Switzerland but no isolates are available for analysis. In addition, we describe two pestiviruses, one a cell culture contaminant and the other isolated from a buffalo, that cluster with a recently proposed novel pestivirus species.

Neurons in monkey visual cortex detect lines defined by coherent motion of dots.

Form perception from coherent motion is an important aspect of vision. Representations of one-, two- and three-dimensional forms have been found at various stages of cortical processing using random-dot stimuli, whereas representations of biological objects like a walking human being concentrate at higher stages of processing. The perception of biological objects can be induced by sparse dot stimuli that consist of a few dots that mark the joints of the human body [G. Johansson (1973) Percept. Psychophys., 14, 201-211]. In the present study, we aimed to investigate whether neurons in early visual areas that respond to bars and edges defined by luminance contrast also signal bar-like objects from sparse dot stimuli. We studied single neurons with rows of 3-24 dots that were either collinear or scattered within a rectangular form. These dots were moved coherently on a uniform or dotted background, and human observers perceived them as rigid rods or other bar-like objects. We found neurons in the visual cortex of the awake, behaving monkey that responded to these stimuli and were sensitive to the orientation of these objects as for conventional bars or edges. Stimulus conditions that failed to induce these percepts in human observers also evoked weaker responses or none in these neurons. We found these neurons with increasing frequency in areas V1, V2 and V3/V3A. The results suggest that the visual cortex not only detects biological objects, but also lines and other bar-like objects from sparse dot stimuli, and that this function evolves at an early stage of processing.

Efficacy of oral vaccination in the final stage of fox rabies elimination in Switzerland.

Subsequent to rabies vaccination campaigns, two well-established methods for the determination of the proportion of vaccinated foxes--the detection of tetracycline (TC) in bones and the detection of virus neutralizing antibodies (VNA) in thoracic fluids--were used and compared. Special emphasis was given to the effect of a new method of bait distribution at the den, which is primarily targeted at young foxes. The overall proportion of vaccinated animals estimated by TC was 60% as compared to 50% by VNA. In young foxes overall, significantly lower proportions of vaccinated animals (58% by TC and 40% by VNA) than in adult foxes (75 and 59%) were estimated with both methods. Low proportions of vaccinated young animals were found after spring (39 and 18%), but also after autumn vaccination (56 and 35%). In contrast, after den vaccination the level of vaccination of young foxes reached that of adult foxes. The theoretical implication of the successful elimination of fox rabies in Switzerland in spite of a relatively low overall proportion of VNA-positive animals is discussed.

[Border disease in a flock of sheep]. / Border Disease in einem Schafbetrieb.

This report describes border disease in a flock of sheep in Switzerland. In April 2001, three ewes in a flock of 41 sheep gave birth to lambs that had generalized tremors and excessively hairy fleece. One of these, a three-week-old female lamb, was referred to our clinic for further diagnostic work-up. The lamb was very nervous, bleated constantly and had generalized muscle tremors, which were more pronounced in the head region. Hind end ataxia was observed, and the lamb was slow to correct its posture when the hind limbs were abducted, adducted or crossed. Blood samples were collected every six weeks to determine antibody titres to pestivirus and for virus isolation via cell culture. A skin biopsy sample was also collected and examined immunohistochemically for pestivirus antigen. Antibody titres in the first tests were suspicious and those of the second were negative. Pestivirus was identified in cell culture, and the skin biopsy sample was positive for pestivirus antigen. Blood samples were collected from all of the ewes and lambs and the buck for virus isolation via cell culture and determination of pestivirus antibody titres. Thirty-one animals were seropositive, six had borderline antibody titres and four were seronegative. Pestivirus was isolated from eight animals, which included the lamb described in this report. Of the virus-positive animals, three were seronegative, three others had borderline titres and two were seropositive. Six of the eight viruses isolated from cell culture were further characterized genetically via retrotranscription and polymerase chain reaction and subsequent sequencing. The phylogenetic analysis revealed that the causative agent was border disease virus. This is the first time that border disease virus has been isolated in Switzerland. The lamb referred to our clinic was observed for three months; it was then euthanatised and a postmortem examination was performed. Immunohistochemical examination of numerous organs revealed pestivirus antigen. The source of infection was though to be infected sheep from another flock, which shared a pasture. All antigen-positive animals were slaughtered.

Phylogenetic analysis of small ruminant lentiviruses from Southern Brazil.

The first lentivirus isolated from sheep in Brazil was analysed phylogenetically. Evolutionary trees of the proviral 597 nucleotide gag and 432 nucleotide pol sequences obtained by the maximum likelihood method demonstrated that the sheep isolate clustered with prototype Maedi Visna virus whereas three lentiviruses isolated from goats in the same geographic region were close to caprine arthritis encephalitis prototypes. A subsequent comparison of sequence data of these viruses with those contained in the EMBL sequence database revealed that, in contrast to caprine prototypic viruses, all prototypic Maedi Visna viruses contain a deletion of six nucleotides in the gag gene resulting in the deletion of two residues in the central region of capsid protein. This deletion may be a useful marker in the analysis of small ruminant lentiviruses, especially when considering possible transmission of lentiviruses between sheep and goats.

The effect of infection with bovine viral diarrhea virus on the fertility of Swiss dairy cattle.

Bovine viral diarrhea virus is a major cattle pathogen with a worldwide distribution. Animals may be infected with BVD virus transiently or persistently. Transient infection leads to protective immunity. Persistent infection is unique because it is associated with an immunotolerance that is specific to the infecting strain of BVD virus. Persistent infection results from viral invasion of fetuses between the second and fourth month of development. Such animals are of prime importance in the epidemiology of BVD because they shed large amounts of virus, and thus serve as a constant source of infection for non-immune animals. Infection of pregnant animals during the first two months of gestation may result in an increased rate of return to estrus. Animals infected in the period of five months to birth may abort or give birth to calves with malformations. The effects of BVD virus on fertility and gestation are well documented from experimental infection. However, much less is known of the extent of losses under field conditions. The main reason for this may be the multitude of other causes of increased return rates and gestation failures. In addition, the incidence of infection with BVD virus may vary over time and depends on management practices. In this study, we investigated the impact of BVD virus on gestation failures under field conditions in a large cattle-breeding area of Switzerland, where no specific measures to control BVD are in effect. Our approach consisted of relating seroconversions to BVD virus with the rate of return to estrus, abortion, and birth of calves with apparent malformations. These parameters of fertility were compared to those of animals immune to BVDV infection due to previous exposure to the virus and animals without seroconversion. Our data show that infection with BVD virus during the first 45 days of gestation did not influence the rate of return to estrus. By contrast, we observed a statistically significant increase in the abortion rate in mid-term gestation (Days 46 to 210) while no such effect was observed in the later stages of gestation. No clinically manifest malformations were observed in the offspring of animals that had seroconverted to BVD virus. In our study population the prevalence of BVDV antibody positive cattle varied only slightly between 78% and 80% over the period of observation. Our data showed that 7% (CI: 2.4-14%) of fetal deaths may be attributable to infection with BVD virus.

Simulation of neuronal responses defining depth order and contrast polarity at illusory contours in monkey area V2.

Neurophysiological, brain imaging, and perceptual studies in animals and humans suggest that illusory (occluding) contours are represented at an early level of visual cortical processing. Comparatively little is known about the mechanisms defining the depth order and the brightness illusion associated with such contours. Baumann et al. (1997) found neurons in area V2 of the alert monkey that signaled not only illusory contours but also the figure-ground direction that human observers perceive at such contours. The majority of these neurons showed this property independent stimulus contrast; a small minority preferred a certain combination of figure-ground direction and contrast polarity at these contours. In this article, we simulate the responses of these neurons by means of a grouping mechanism that uses occlusion cues (line-ends, corners) to define figure-ground direction and contrast polarity at such contours.

Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Both noncytopathic (ncp) and cytopathic (cp) biotypes of BVDV can be isolated from persistently infected cattle suffering from the lethal mucosal disease. The cp biotype correlates with the production of the NS3 nonstructural protein, which in the corresponding ncp biotype is present in its uncleaved form, NS23. Previously, we have shown that cp but not ncp BVDV induces the formation of alpha/beta interferons in bovine macrophages. In this study, we demonstrate that ncp BVDV inhibits the induction of apoptosis and the expression of interferon alpha/beta by poly(IC), a synthetic double-stranded RNA (dsRNA). Inhibition was observed only in cells which had been infected with ncp BVDV at least 12 h prior to the addition of dsRNA, which indicates that expression of viral proteins is necessary for the ncp virus to inhibit the effects of poly(IC). Additional experiments using transfected poly(IC) showed that ncp BVDV interfered with the intracellular action of dsRNA rather than with its uptake into the cells. Infected cells were not resistant to induction of apoptosis by actinomycin D or staurosporine, which suggests that ncp BVDV may specifically interfere with signaling through dsRNA. Interference with the innate antiviral host responses may explain the successful establishment of persistent infection by ncp BVDV in fetuses early in their development.