Preferred citation: Anipedia, www.anipedia.org: JAW Coetzer and P Oberem (Directors) In: Infectious Diseases of Livestock, JAW Coetzer, GR Thomson,
NJ Maclachlan and M-L Penrith (Editors). E J Dubovi, Bovine viral diarrhoea and mucosal disease, 2018.
Bovine viral diarrhoea and mucosal disease

Bovine viral diarrhoea and mucosal disease

Previous authors: L N D POTGIETER

Current authors:
E J DUBOVI - Professor of Virology, MS, PhD, Animal Health Diagnostic Center, 240 Farrier Road, Ithaca, New York, 14853, USA

Introduction

Bovine viral diarrhoea (BVD) and mucosal disease (MD) are two of several disease syndromes in cattle caused by bovine viral diarrhoea virus (BVDV). The primary economic impact of BVDV is mainly at the level of the bovine embryo or foetus, resulting in embryonal/foetal death, teratogenesis, persistent infection or inapparent infection and post-natal poor performance. A late consequence of some foetal infections is the birth of calves with persistent infections that subsequently result in the development of MD, a severe form of BVD with an unusual aetiology. Primary post-natal infections in cattle usually result in a mild form of BVD characterized by inappetance, fever and leukopenia. However, instances of severe and sometimes fatal BVDV-induced disease of cattle showing fever, diarrhoea, respiratory disease and/or a generalized haemorrhagic syndrome have been reported. Several detailed reviews on various facets of the disease have been published.6, 17, 23, 113, 138, 178, 183, 210, 220

Bovine viral diarrhoea was first described in 1946 in the USA by Olafson and co-workers.163 The disease was characterized by abortions, fever, inappetance, diarrhoea, and ulceration of the gastrointestinal tract. Morbidity was high and the mortality in outbreaks was 4 to 8 per cent. A syndrome consistent with mucosal disease, initially referred to as ‘X disease’, was first recorded in Canada, also in 1946, by Childs.58 Ramsey et al. coined the term ‘mucosal disease’ in 1953 based on their observations of the nature of the disease and lesions in  Iowa, USA.186

Aetiology

Bovine viral diarrhoea virus is a member of the Pestivirus genus of the family Flaviviridae.140 (See General Introduction: Flaviviridae). Currently, four species of pestiviruses are recognized by the International Committee on Taxonomy of Viruses; BVDV-1, BVDV-2, border disease virus and classical swine fever/hog cholera virus. (see Border disease and Classical swine fever) The number of species within the genus Pestivirus will certainly increase as “atypical” pestiviruses of swine and wild ungulates are formally classified.  A novel BVDV virus referred to as Hobi or BVDV-3 may generate a third species within the bovine pestivirus group.208, 210 Many different genotypes (subtypes) have been reported within the species, but their biological relevance has yet to be determined.

Bovine viral diarrhoea virions are spherical particles approximately 50 nm in diameter, with a tightly adherent envelope containing glycolipids.140 The virions are difficult to detect by negative-stain electron microscopy, but roughly spherical particles with a 20 to 25 nm core are readily detectable in infected cells where the virus replicates entirely within the cytoplasm.101 Because the virus has a lipid-rich envelope, it is susceptible to most common disinfectants.140

The BVDV genome consists of a single strand of RNA with positive polarity and contains one large open reading frame (ORF) that encodes a polyprotein of approximately 3900 amino acids (Figure 1). This open reading frame is preceded by a short untranslated region (UTR) that functions to initiate protein synthesis.176 Protein maturation is the consequence of co-translational and post-translational cleavage of the polyprotein by viral and host cell proteases. Except for the first protein encoded by the BVDV genome (Npro — a viral protease), the structural proteins are encoded by the first third of the genome (5’ end), whereas the non-structural proteins are encoded by the last two-thirds of the genome.220

The structural proteins include the internal capsid protein (C) and the virion surface envelope proteins. Erns (or E0) is only loosely associated with the virion and has intrinsic RNase activity. This RNase activity was reported to inhibit interferon responses in infected cells.119, 144 E1 is a transmembrane glycoprotein that forms a dimer on the virion surface with glycoprotein E2 (Figure 1). The latter, the major virion glycoprotein, is the principal target of virus-neutralizing antibodies, is the receptor-binding protein of the virus, and determines host-range.

The non-structural (NS) proteins (p7, NS2-3, NS2, N3, NS4A, NS4B, NS5A, and NS5B) are not incorporated into mature virions and function in viral replication/RNA synthesis and viral morphogenesis.  NS5B is an RNA-dependent RNA polymerase, but replication of the RNA genome requires NS3-NS5B proteins acting in concert.  As seems the general rule with viral proteins, multiple functions are present within a single viral protein.  So, NS3 has a serine protease domain, helicase activity and NTPase activity.  In addition to enzymatic activities, NS3 either alone or in concert with uncleaved NS2-3 protein, is essential for viral morphogenesis. Npro as the first protein translated is an auto-protease that releases itself from the next protein in the ORF, capsid protein C.  Npro is not essential for virus replication in vitro, but it does have the ability to antagonize the induction of an IFN-1 response, albeit weakly.   In conjunction with Erns, Npro may play a role in suppressing the antiviral responses in infected animals.220

Figure 1 The structural map of the bovine viral diarrhoea virus genome. It consists of single-stranded RNA with positive polarity. Except for the untranslated (UTR) regions at either end, the genome is translated as one open reading frame. The various viral proteins (Npro, C, Erns, E1, E2, NS2-3, NS4A, NS4B, NS5A, NS5B) produced by enzymatic cleavage of a polyprotein

Two biotypes of BVDV are identifiable in the laboratory: non-cytopathic isolates that do not obviously affect the integrity of cultured cells and cytopathic isolates that induce cytopathic effects and the death of infected cells. Genotypes are member variants of each of the BVDV species80, 194 and non-cytopathic and cytopathic isolates exist within each genotype. Genotypes are usually classified by the base sequence of the relatively conserved, 5’ untranslated region of their genomes,194 but genes such as NS3 have also been used.  Genotyping schemes are not universally accepted and their value may only be in an epidemiological context as no studies have shown unique biological relevance to a given genotype. Severe primary postnatal infections due to BVDV-2 have been recognized only in recent years and this has led to speculation that the BVDV- 2 isolates are more virulent than the BVDV-1 isolates.33, 87, 162 Although BVDV-2 virus was initially identified in North America, the evidence is that both BVDV species have been co-circulating in cattle throughout the world.50, 213, 248 BVDV-2 viruses have been identified in southern Africa along with multiple subtypes of BVDV-1. The prevalence rate of the two species is similar to what has been reported in the US - ~80 per cent BVDV-1 and 20 per cent BVDV-2.236

Although all pestiviruses are antigenically related, virus neutralization data reported by several investigators suggest that BVDV of different genotypes (UTR-based) may have some antigenically (neutralization) unique epitopes.30, 59, 71, 166 For this to be universally true would require that specific characteristics of the UTR region be associated, in some way, with sequences in the translated region of the virus.  The fact that viable hybrid viruses can be constructed indicates that UTR-defined genotypes may not correlate with antigenic types.

Antigenic analyses of BVDV isolates based on virus neutralization with polyclonal and monoclonal antisera have verified that significant variations exist in some epitopes involved in viral neutralization.30, 59, 82, 167, 171, 238, 250 Most of the latter appear to be present in the major glycoprotein (E2) on the surface of the virus. Genomic regions encoding two amino acid stretches within this protein may be the most variable region of the BVDV genome. The BVDV genome, as with most RNA viruses, is highly variable and viruses such as these are considered by molecular biologists to be a ‘quasispecies’.77 This variability contributes to the complexity of classification of BVDV isolates.71, 85, 171, 238, 250 The BVDV quasispecies therefore should be viewed as genotypes consisting of families of antigenically related variants and that variants within each genotype may be more related to one another than to variants in another genotype. It is probable that the neutralizing antibody response of the host contributes significantly to the evolution of surface glycoprotein epitopes of the virus. It is not surprising that a virus with such a plastic genome generates variants with wide differences in antigenicity, virulence and perhaps tissue or cell tropism. The recognition of unique antigenic fingerprints of the E2 glycoprotein in individual isolates of BVDV is central to the understanding of the pathogenesis of MD (see Pathogenesis).

Most field isolates of BVDV are non-cytopathic and have an ‘intact’ NS2-3 gene;77, 169 the molecular mass of the gene product being 125 kD. Cytopathic BVDV isolates are frequently isolated from animals suffering from MD and they produce, in addition to the NS2-3 gene product, at least two additional cleavage products, i.e. a 54 kD protein (NS2) and an 80 kD protein (NS3). The latter is considered a molecular marker for cytopathic BVDV isolates and the cytopathic effect may be due to the generation of NS3 within infected cells which leads to a significant increase in RNA synthesis that triggers an apoptotic response.77, 78, 220 The production of NS2-3 cleavage products appears to be the consequence of recombinational events during viral replication. These include the insertion of cellular RNA sequences into the NS2-3 gene or duplicated viral genes that become inserted within this region or in neighbouring regions of the genome.14, 91, 92, 149, 184, 196, 218, 219 However, recombination resulting in genetic insertions in non-cytopathic isolates do not always cause biotypic conversion.197 In some instances, the mutant cytopathic viruses result from deletions or point mutations instead of recombination.127, 156 Some of these isolates are defective and are dependent for replication on the presence of a non-cytopathic “helper” virus.

Epidemiology

Bovine viral diarrhoea virus infections in cattle have been reported from most regions of the world. Evidence of such infections in southern Africa was first reported in the early 1970s225, 226, 228 and they appear to be widespread in the cattle populations of the region.12, 70, 226, 228

Natural pestivirus infections and disease occur, apart from cattle, also in sheep, pigs and wild boar, goats, a wide range of captive and free-living ruminants (virtually all even-toed ungulates) and perhaps free-ranging rabbits.70, 93, 96, 104, 117, 251 There are very few reports on the isolation of pestiviruses from non-domesticated ruminants79, 95, 107, 138, 158 but serological surveys have strongly implicated pestivirus infections in members of the Camelidae, Cervidae, Antilocapridae and Bovidae.3, 70, 94, 117, 251

The susceptibility of various animal species to BVDV infections has been reviewed by Løken.138 Evidence is that BVDV, or closely related viruses, cause frank disease in a number of wildlife species including giraffe (Giraffa camelopardus), African buffalo (Syncerus caffer), eland (Taurotragus oryx), Indian buffalo (Bubalus bubalis), roe deer (Capreolus capreolus) and red deer (Cervus elaphus).95, 107, 138, 158 Some African buffalo and blue wildebeest (Connochaetes taurinus) herds suffer high infection rates.117 An antibody prevalence rate of over 40 per cent among kudu (Tragelaphus strepsiceros), eland and giraffe in Namibia was reported.70 In Zimbabwe, prevalence is high among eland, nyala (Tragelaphus angasii) and bushbuck (Tragelaphus scriptus).3Some evidence suggests that BVDV isolates infecting free-ranging ungulates are distinct genotypes of the virus.13, 89, 95, 210.

The most important sources of BVDV in nature are persistently infected (PI) bovids that result from in utero infection prior to the maturation of the foetal immune response (see Pathogenesis).113, 114, 118, 222  The blood of these animals may have as much as 107 cell culture infectious doses of virus per millilitre.40 High virus levels are usually present in such animals in nasal secretions, saliva, tears, semen, and milk. Susceptible animals in close proximity with a persistently infected bovids rapidly become infected.113, 118, 222 Therefore, herds containing persistently infected animals have a high proportion of animals that are antibody-positive. Herds with few serologically positive animals are at risk of infection with the consequent risk of persistently infected calves being born.113, 114, 222

Animals undergoing primary, postnatal infections are transiently infected and eventually clear the virus but also may be a source of the virus that could infect animals in contact with them, especially in herds with low levels of herd immunity.113, 118 The secretions and excretions of such animals usually contain infectious virus from four to ten days post- infection.

The gross infectivity in secretions and excretions of transiently infected cattle are several thousand times less than those of persistently infected animals. The virus therefore spreads with much lower efficiency from transiently infected animals than from persistently infected individuals (low virus levels plus shorter shedding period).

Both vertical and horizontal transmission of BVDV may occur; the virus is therefore perpetuated by cycles of vertical and horizontal transmission.9 Bovine viral diarrhoea virus crosses the placenta efficiently to infect embryos or foetuses.18 In utero infections occur consistently when naive pregnant heifers/cows are infected with the virus. The embryo/ foetus may be protected from infection in cows that have been immunized by previous natural infection or vaccination.159  Persistently infected cows and heifers always deliver persistently infected calves. This promotes the establishment of family lines of persistently infected animals for several generations, resulting in unusually high prevalence of infected animals in certain herds.27

Although BVDV infects many animal species, and interspecies transmission of the virus has been documented, in most instances cattle serve as the source of the virus.120, 138, 223, 224 Animals other than cattle do not appear to be important sources of infection for cattle populations. Persistent BVDV infections have been recorded primarily in cattle and, to a lesser extent, in sheep and pigs, but MD has been described in some wildlife species which suggests that persistent infections are possible in at least some of these species.138 Persistently infected white-tailed deer have been found in natural settings.54

Semen from persistently infected bulls may contains high concentrations of BVDV and may result in poor conception rates in susceptible heifers/cows after breeding or insemination. Following insemination, naive heifers/cows usually become transiently infected and, in some instances, have produced persistently infected calves. Only a small proportion of sero-negative heifers inseminated with semen from transiently infected bulls usually become infected, perhaps reflecting the low virus levels in the semen of transiently infected animals.124, 125 Although foetal infections may occur occasionally, significant reduction in conception rates when semen from transiently infected bulls is used are rare. However, secondary cycles of transmission from transiently infected inseminated cows may occur, reducing overall reproductive performance for the herd.146  Semen should consequently only be sourced from suppliers with an active BVDV management program.

Vertical transmission will always occur after embryo transfer if the recipient is persistently infected.16, 234  For that reason, screening embryo recipients for their infection status should be a standard operating procedure.  Bovine viral diarrhoea virus is also present in the ovaries of persistently infected cows.90, 103, 233, 234 Use of feeder cells (oviduct epithelial and granulosa cells) from persistently infected animals presents substantial risk of transmission to bovine embryos during embryo transfer procedures.35 In addition, adventitious contamination of bovine oocytes or embryos during in vitro fertilization procedures (usually from commercial foetal calf serum) may result in transmission of the virus.97, 234  Embryos, at least to the blastocyst stage, may not be affected by the virus if the zona pellucida remains intact.232, 233, 240

Bovine viral diarrhoea virus   is transmitted horizontally by direct or indirect contact. The primary mechanism of postnatal BVDV transmission is direct contact between persistently infected and susceptible animals.7, 222 Aerosol transmission over short distances appears likely but the probability of virus transmission occurring decreases rapidly as the distance between animals increases. The potential for indirect transmission is dependent on the stability of the virus outside the host. Laboratory studies suggest that the virus is stable below 10° C and over a wide pH range (3 to 9).113 The virus may survive in natural environments for three hours at 35 °C, three to seven days at 20 °C and three weeks at 5 °C.113 Contamination of feed and water with saliva from a persistently infected animal is a potential source of virus for horizontal transmission.  Mechanical transmission of the virus among cattle by biting insects has been reported by Tarry et al.113, 114 Iatrogenic infections may occur as a result of contaminated hypodermic needles, nose tongs, instruments used for ear tagging and castration operations, as well as during a series of rectal examinations. As with vertical transmission, horizontal transmission may occur via semen or during embryo transfer procedures. Other means of indirect transmission by fomites, such as contaminated clothing, are thought to be possible. Vaccines contaminated with adventitious BVDV have been the source of infections in the past.8

Bovine viral diarrhoea virus  often gains entrance into a herd by the introduction of a persistently infected animal or a pregnant animal carrying a persistently infected foetus. The spread of infection in susceptible herds in such instances is usually not explosive, but most contact animals become antibody-positive within a few months. Slower transmission may occur in the absence of persistently infected animals within a herd. This is probably due to ongoing transient primary postnatal infections which generate lower amounts of virus for a short period of time.  The presence of persistently infected animals usually results in herd-specific BVDV isolates whereas significant variation of isolates often exists within herds where persistently infected animals are not present.108

The sequence of events in a susceptible herd after introduction of a persistently infected animal usually follows a predictable pattern.150 Initially, clinical signs of primary postnatal infections may develop, which are followed immediately by repeat breeding for a few months and then by abortions for several months. Calves with congenital defects may subsequently be born followed by the birth of persistently infected animals. The latter usually arrive five to nine months after the first clinical signs of transient infection occurred. A few calves may have congenital defects, and some may have increased susceptibility to other diseases. However, it is rare to encounter all the clinical manifestations of the disease in the same herd. Factors contributing to this variation include the nature of the transmission pattern (eg. route of infection and level of virus), herd immunity at the time of infection, pregnancy status of animals at the time of infection, and the virulence of different BVDV isolates. For example, initial transient infections may be mild and go unnoticed and the first manifestation of the disease may be reproductive disorders. The first abortions are unlikely to occur until three to four weeks after the introduced of the virus into a herd.

Pathogenesis

Primary postnatal infections

The virus first replicates in the naso-pharyngeal mucosa and, to high titres, in the tonsils.87, 142, 214, 247 It then spreads to the regional lymph nodes followed by cell-associated (leukocyte) dissemination throughout the body. The highest levels of virus usually develop in the tonsils, thymus and ileum.

The manifestations of most primary postnatal BVDV infections  are characterized by mild fever only, leukopenia and, occasionally, diarrhoea.247 Several BVD outbreaks reported during the last decade in North America and the UK, however, have been severe (see Clinical signs).168 Pellerin et al. concluded that some highly virulent BVDV isolates belong to a specific and distinct genotype (BVDV-2 or type 2).168 It appears that these isolates are more likely to target thrombocytes than classical BVDV-1 isolates. However, some BVDV-1 isolates may also have the capacity to impair platelet function and resulting in extensive haemorrhage in infected animals. Genotype alone does not confer virulence. Experimental evidence suggests that isolates vary in their virulence and capacity to induce thrombocytopenia.241, 242 Diarrhoea, fever and lower respiratory tract disease with leukopenia, anaemia, thrombocytopenia and a mortality rate as high as 10 per cent were commonly encountered. In some instances, the infected animals manifested extensive haemorrhages, a consequence of the severe thrombocytopenia (see Pathology). Megakaryocytes and lymphocytes constitute important targets of the virus.241 The virus causes necrosis of these cells and impairs the function of those that survive infection.242 Osteopetrosis, the apparent consequence of a transient interruption of endochondral ossification, has also been associated with some of these infections.211 It is of interest to note that Olafson and his co-workers in the original description of the disease recorded the presence of haemorrhages in various tissues.163

Some isolates of the virus (including those used in vaccines) replicate in the ovaries and the potential exists for ovarian dysfunction and impaired reproduction as a consequence.102 The virus also has been detected at relatively high concentrations in the islets of Langerhans in the pancreas and in the pituitary gland of experimentally infected animals.214

Bovine viral diarrhoea virus infections have been associated with lower respiratory tract disease.178 The degree to which this virus contributes to the prevalence of such disease remains a contentious topic among veterinary researchers. The results of a few studies suggest that some BVDV strains may constitute a primary pneumopathogenic agent.147, 180 Experimental reproduction of lower respiratory tract disease with the virus appears to be dose dependent.182 Some isolates are able to elicit the primary respiratory tract lesions of interstitial and bronchopneumonia and tracheitis.147, 178, 180 Nevertheless, it appears that the major factor in the development of BVDV-induced respiratory tract disease is the ability of the virus to induce immunosuppression and thereby enhance disease caused by opportunistic pathogens (see Pathogenesis: Immunosuppression).

Prenatal (in utero) infections

Most- but not all-transplacental infections are caused by non-cytopathic isolate. The outcome of intra-uterine infections with BVDV is determined primarily by the stage of gestation and the virus isolate.18, 51, 76, 145, 146 The virus affects fertility by several mechanisms. The semen quality of infected bulls may be reduced dramatically as a result of a decrease in sperm density and motility, and an increase in sperm abnormalities. The virus is capable of causing prolonged oophoritis and may be a major cause of repeat breeding.103 Sero-negative heifers inseminated with contaminated semen or those exposed to the virus intranasally just prior to breeding may experience considerably reduced conception rates.124  The virus may also interfere with fertilization but it does not have the ability to penetrate an intact zona pellucida. However, in vitro studies suggest it remains associated with the zona pellucida where it may serve as a source of infection of the blastocyst when the zona pellucida disintegrates just prior to implantation.97, 98, 232, 240

During the first trimester of pregnancy, embryos and foetuses are highly susceptible to the virus. Susceptibility is positively correlated with gestational age and is linked to the ontogeny of the immune response which develops in a stepwise manner.18 Transplacental infection of foetuses of up to 125 days of gestation may result in their death, resorption, expulsion between several days to several months later, or the generation of a persistently infected foetus.222

Bovine viral diarrhoea virus is an important teratogen in calves and defects may develop following transplacental infections during early to mid-gestation (i.e. about 60 to 150 days). This period of foetal development corresponds to the final stages of organogenesis and development of the foetal immune system. An inflammatory response may occur, but the primary mechanisms underlying the virus-induced lesions include inhibition of cell growth and/or cell differentiation, and direct cell lysis. The congenital effects (see Clinical signs) are numerous and include lesions/defects of the nervous system, eye, immune system, integument, and musculo-skeletal and respiratory systems. Diabetes mellitus in young calves has been attributed to in utero BVDV infection,216, 217 which is probably the result of infection of the islets of Langerhans as described in experimentally infected calves.214 The foetal thyroid also appears to be a target of the virus and subsequent impaired thyroid function may be the cause of unthriftiness in infected calves.133 Furthermore, the virus appears to have the capacity to infect the pituitary gland, another potential cause of unthriftiness in calves.214

Transplacental infections by BVDV during the third trimester of pregnancy in cattle normally do not result in foetal disease and are analogous to primary postnatal infections. These foetuses mount relatively normal immune responses and are born with circulating antibodies to the virus.  While not common, abortions due to a BVDV infection can occur late in gestation.

Some infections with non-cytopathic BVDV of the foetus before the development of immuno-competence may result in specific immunotolerance to the virus.60, 145 Infections that result in persistently infected foetuses occur between 60-125 days of gestation. Immunotolerance results in the birth of persistently infected calves that are viraemic and continuously shed virus.60  Persistently infected cattle are immunotolerant only to the infecting BVDV isolate; therefore they are immunocompetent, at least to a degree, with respect to other antigens, including those of other pathogens and  unique epitopes of heterologous BVDV isolates.21, 28 Consequently, although persistently infected animals are usually sero-negative, some may be sero-positive to BVDV strains in modified-live vaccines used in herd control programs.

Colostrum-derived antibodies decline more rapidly in persistently infected animals than in normal animals primarily due to removal of the antibodies through binding to viral antigens. Some persistently infected animals are stunted and grow slowly. The latter condition has been associated with a deficiency of thyroid hormones.133 Persistently infected animals are at risk of developing other diseases, such as pneumonia and enteritis, and many may die during their first year of life (see below).221, 222 A small number of persistently infected animals appear normal but subclinical glomerulonephritis and encephalitis have been described in some of these ‘healthy’ animals.65 In addition, significant morphological changes may occur in the ovaries of persistently infected cows, which suggests that their reproductive performance may be impaired.103 All persistently infected animals are at risk of developing MD.

Mucosal disease is a severe, usually fatal condition that may have an acute or a protracted course. It occurs when a persistently infected animal is “super infected” with a cytopathic virus that usually has a close antigenic relationship to the resident non-cytopathic virus.24, 28, 29, 46, 115, 155, 198, 219 The cytopathic virus is most commonly derived from the resident non-cytopathic virus in the persistently infected animal by molecular rearrangement of its genome (see Aetiology)77 but its origin can be external such as occurs in vaccine-associated outbreaks. The mutational event responsible for changing the non-cytopathic biotype of the virus in a persistently infected animal to a cytopathic biotype does not affect antigenicity.59 Thus, the new biotype is homologous antigenically to the resident non-cytopathic biotype and is not cleared by the immune system. One hypothesis is that cytopathic BVDV then spreads throughout the host causing, among other lesions, progressive depletion of gut-associated lymphoid tissue and necrosis of its overlying mucosa. Initially, in animals suffering from MD, cytopathic virus is present most consistently in the tonsils, lymph nodes, Peyer’s patches, and lymphoid nodules of the large intestine.136, 139 In the late phase, a diffuse distribution of the virus in the intestinal epithelium appears to correspond with the advent of clinical disease. Cellular destruction by cytopathic BVDV appears to be mediated by programmed cell death (apoptosis).1, 111, 129, 252

Usually MD has a rapid onset of severe BVD with a short period of disease before death (early-onset MD). Occasionally, the development of MD in persistently infected animals is delayed for several weeks or months (late-onset MD).193

Late-onset MD is delayed because a more convoluted and slower process is involved through which the cytopathic virus becomes established in persistently infected animals. It appears to be the result of external superinfection either with a closely related or even with a heterologous cytopathic virus.48, 91, 92, 193 The superinfecting cytopathic virus in such cases therefore differs antigenically from the resident non-cytopathic virus of a persistently infected animal. The superinfecting virus replicates and is cleared by the animal’s immune response but contributes to the creation of a new cytopathic virus that cannot be cleared, resulting in the eventual development of delayed onset MD. The new cytopathic mutant may originate by recombination, resulting in a hybrid virus that is cytopathic but has the genetic sequences encoding neutralizing antigens of the resident non-cytopathic virus.

Immunosuppression

Dinter and Bakos in 1961 described severe respiratory tract disease (Umea disease) in cattle in Sweden which was the likely consequence of a synergistic infection of parainfluenza 3 virus and BVDV.75 Borgen and Dinter subsequently reported the occurrence of Umea disease in Denmark.36 Considerable evidence has accumulated that BVDV may be a pivotal component in multiple-aetiology infectious diseases.177, 178, 192 Bovine viral diarrhoea virus in association with other potential pathogens, is frequently encountered in diseased tissues,192, 215 and may be the common denominator in the pathogenesis of such diseases.215  It is recovered with high frequency from diseased respiratory tract tissues infected usually with Mannheimia (Pasteurella) haemolytica type 1 and/or bovine herpesvirus 1 (BHV-1), the cause of infectious bovine rhinotracheitis.249 The possible synergistic involvement of BVDV in multiple infections is usually unrecognized because ‘classical’ BVDV lesions are absent. Bovine viral diarrhoea virus was the virus most often recovered from pneumonic lungs (21 per cent) of Texan feedlot cattle.189 It was usually associated with M. haemolytica type 1 infection as well as with severe pneumonia unresponsive to antibiotic therapy. It has been hypothesized that ‘subclinical’ BVDV infection may be a significant contributor to the incidence of severe respiratory tract disease in dairy cattle.178 Other infectious diseases that BVDV may exacerbate include actinomycosis, papular stomatitis, enteritis caused by Salmonella serovars, Escherichia coli infections, babesiosis, acute helminthosis, metritis and mastitis.20, 47, 170 In most instances of case reports involving BVDV’s enhancement of other pathogens, no attempt was made to distinguish between acute BVDV- and persistent BVDV infection.

Transient leukopenia occurs in most cattle infected with BVDV, and lymphoid depletion is a frequent post-mortem finding. Cattle with primary postnatal infections have a reduced numbers of B- and T-lymphocytes, the percentage of T-lymphocyte subsets, and neutrophils.26, 86 A decline may occur in BoT4+ (helper), BoT8+ (cytotoxic/suppressor) and gamma delta+ lymphocyte subsets but not in non-T, non-B (null) lymphocytes or monocytes in these animals.86 In addition, the numbers of cells expressing major histocompatibility class O antigens may be affected.86 As more detailed measurements are made of the innate and adaptive immune responses following acute BVDV infections, the picture that emerges is one of a general detrimental impact on immunocytes.55

The foregoing observations on enhancement of other pathogens are supported by experimental data, e.g. for the synergistic interaction between BVDV and BHV-1 in calves reported in 1984.181 Incipient BVDV infection impaired the ability of calves to clear BHV-1 from the lungs and to contain the latter virus at the local infection site. Similar results for bovine respiratory syncytial virus (BRSV) infections have been reported.88, 175 It was observed  that BVDV and BRSV may act synergistically in eliciting bovine respiratory tract disease.227 Experimental evidence  indicated that BVDV also greatly enhances respiratory tract disease induced by BRSV.42, 121 The mechanism for this enhancement may be suppression of the IFN-1 pathway signalling by BVDV.2 Additional experimental evidence to support this hypothesis includes the observations that BVDV infection in calves promoted the dissemination of endogenous bacteria, resulting in a transient bacteraemia coinciding temporally with leukopenia and suppression of lymphocyte stimulation by phytohaemagglutinin.190

The potential for synergism between BVDV and other pathogens is also supported by accumulating laboratory evidence which indicates that BVDV causes a profound and broad-spectrum deficit in the immune response of cattle.4, 5, 55, 74, 106, 122, 130, 131, 141, 152, 153, 200, 231 The virus has an affinity for immune effector cells and a consequence of infection is the destruction of some of these cells.231 Impaired function of surviving cells, however, is another consequence of infection that may be a more important cause of the ensuing immunosuppression induced by this virus.4, 44, 56, 122, 201-203  Most viruses have mechanisms to suppress the innate immune responses.  Bovine viral diarrhoea has two genes, Npro and Erns, that inhibit the up-regulation of the interferon response gene network and in doing so may enhance the ability of other co-infecting pathogens to cause overt disease.2

Respiratory tract disease

As noted previously, BVDV may be an important bovine respiratory tract pathogen.147 Its primary role in respiratory tract disease may be its capacity to facilitate and enhance secondary infections by interfering with the immune response  to other potential pathogens.83, 88, 99 Bovine viral diarrhoea  may impair bacterial clearance from the respiratory tract of  calves, promote dissemination of bacteria within the lower respiratory tract and enhance the disease process produced by M. haemolytica. Several investigators have described the deleterious effects of BVDV on peripheral immunocompetent cells and on antibody responses, but it has also been determined, by quantifying cells in bronchoalveolar lavage fluids, that infected calves have reduced numbers of pulmonary lymphocytes and neutrophils.235 This virus also replicates in and destroys, or impairs the function of, bovine alveolar macrophages, cells that have a crucial role in the defence of the lung against microorganisms.244

Clinical signs

Bovine viral diarrhoea virus infections in cattle may result in one of three well-defined disease syndromes: BVD (or primary postnatal infections), MD, and embryonal/foetal disease.6 Many host, viral, environmental and epidemiological factors as described in the section on Pathogenesis determine the nature of the disease that develops in animals.

Primary postnatal infections

Subclinical infections or mild BVD

Between 70 to 90 per cent of cattle infected with BVDV do not develop obvious clinical signs. Close observation may reveal leukopenia, slight pyrexia, mild gastro-intestinal signs, and, in some cows, a drop in milk production.

Animals that contract subclinical infections or mild disease may be viraemic from 4 to 15 days after initial infection and develop circulating antibodies to BVDV within two to four weeks after exposure. The role of primary postnatal infections in enteric or respiratory tract disease in young calves is poorly defined. Some instances of diarrhoea and/or respiratory tract disease in young calves associated with BVDV may be the result of persistent infection. Evidence indicates that colostrum-derived antibody provides protection against systemic and respiratory infections caused by BVDV, but calves in group housing with a persistently infected animal may show significant morbidity six to eight weeks after calving as the colostral antibodies decline.

Bovine viral diarrhoea

If frank clinical signs are evident, the disease usually is referred to as BVD. Outbreaks of watery diarrhoea of varying severity may occur in susceptible herds, usually involving 6- to 12-month-old animals. Typically, mortality is negligible but up to 8 per cent has been reported. Morbidity rates may be as high as 30 to 90 per cent. Clinical signs may be evident for five to seven days and include a transient fever, depression, anorexia, hyperpnoea, oculo-nasal discharges, salivation, oral erosions and ulcerations, diarrhoea and, in cows, decrease in milk production.

Severe bovine viral diarrhoea/haemorrhagic syndrome

A severe and sometimes fatal form of acute BVDV infection usually caused by non-cytopathic viruses h occurred in dairy cattle and veal calves in the UK and North America. The disease is characterized by sudden onset of depression, pyrexia, leukopenia, thrombocytopenia, diarrhoea, nasal discharge, salivation, oral ulcers and decreased milk production.87 Morbidity rates as high as 40 per cent and mortality rate of 10 to 25 per cent have been reported. In some outbreaks, pneumonia and/or a haemorrhagic syndrome are features of the disease. The latter is characterized by bloody diarrhoea, epistaxis, widespread petechial and ecchymotic haemorrhages, bleeding from injection sites and, occasionally, hyphema. Outbreaks are often associated with recent introduction of animals into a herd and inadequate vaccination programmes. Most of these outbreaks have been associated with BVDV-2 isolates although the US isolates were a unique subset of a BVDV-2 variants that became widely disseminated resulting in an over-emphasis of BVDV-2 as the cause of severe disease.195 With the availability of genotyping more cases of severe BVD associated with BVDV-1 have been identified.

Respiratory disease and miscellaneous diseases

Respiratory tract disease is frequently associated with infections with this virus178 (see Pathogenesis).

Embryonal/foetal disease

Fertility

Artificial insemination with contaminated semen or natural service by BVDV-infected bulls may result in poor pregnancy and conception rates, embryonal/foetal death and impaired embryonic development (see Pathogenesis).

Abortions

Transplacental infections may cause foetal death and subsequent expulsion of stillborn or mummified foetuses several days to several months after infection (see Pathogenesis). The abortion rate varies but is usually low (2 to 7 per cent) although different rates (0,1 to 27 per cent) have been published.6 Infections in early gestation are more likely to cause abortions but many factors influence that outcome, not the least of which is virus strain.  Late-term abortions are less frequent.

Congenital defects

Congenital defects may occur in foetuses infected at 60 to 150 days of gestation.17, 110 The virus targets the central nervous system and ocular tissues in particular. Defects include cerebellar hypoplasia (Figure 2), microencephaly, hydranencephaly, porencephaly, hydrocephalus, cystic septum pellucidum, dysmyelination, spinal cord hypomyelinogenesis, chorioretinopathy and dysplasia, cataracts, interstitial keratitis, microphthalmia and optic nerve neuritis and atrophy. Other congenital defects caused by the virus are thymic hypoplasia, hypotrichosis, alopecia, curly hair coat, brachygnathism, arthrogryposis, skeletal defects due to deranged osteogenesis, growth retardation and pulmonary hypoplasia.

Calves with cerebellar hypoplasia or other brain anomalies have difficulty in standing and moving. They often stand with splayed legs and may have tremors. The latter may be the consequence of hypomyelogenesis. Cataracts, blindness and corneal opacity are common in such calves. Stunted calves are the consequence of low birth/organ weights and growth retardation and may include relatively short tibial diaphyseal and metaphyseal lines.

Persistently infected calves resulting from infection during the first 60-125 days of gestation may appear normal but some are unthrifty and a high percentage die in their first year as a consequence of non-BVDV related illness.221, 222 These animals have an increased risk of contracting other diseases such as pneumonia and enteritis (see Pathogenesis: Immunosuppression). Close inspection of persistently infected calves often reveals areas of alopecia, hyperkeratosis or eczema in the neck or back region.

Mucosal disease

Two forms of MD have been identified, i.e. acute and chronic forms, the latter with a protracted course. The disease is generally sporadic occurring most frequently in 6 to 24 month-old animals. Low morbidity rates (less than 5 per cent) characterise this form of the disease although ‘outbreaks’ affecting a relatively high proportion of animals have been reported. The case fatality rate approaches 100 per cent. Occasionally, persistently infected calves develop transient signs of MD; this suggests that recovery is possible but appears to be rare.

Acute MD is characterized by pyrexia, depression, weakness, anorexia, profuse watery diarrhoea which may be haemorrhagic, dehydration, acidosis and emaciation. Erosions, ulcerations and large areas of mucosal necrosis involving the lips, gingival margins, tongue, dental pad, commissures of the mouth and hard palate are generally present. Similar lesions also may develop on the muzzle, in the nasal cavity, on the vulva and skin of the teats. Salivation and a mucopurulent ocular discharge of varying severity are common signs. Corneal oedema is sometimes present. The faeces are watery and frequently foul-smelling and may contain necrotic debris, blood and fibrinous casts. Diarrhoea usually develops two to three days after the onset of clinical signs but, in per-acute cases, the animal may die before that is evident. The course of acute MD varies from two to 21 days. Severe leukopenia, accompanied by neutropenia (without a left shift) and lymphopenia may be evident in the early stages of the disease. Thrombocytopenia is often also present. Infection with opportunistic pathogens is common in these animals (see Pathogenesis: Immunosuppression). There is a report on the occurrence of diabetes mellitus in animals suffering from MD.216

The clinical signs of chronic MD are similar to those of the acute disease but less severe and its course protracted (up to 18 months). The disease is characterized by anorexia, progressive emaciation, and continual or intermittent diarrhoea and oculo-nasal discharges. Chronic bloat may be a feature of the disease. Skin lesions are common in chronically affected animals, usually manifested as areas of alopecia and hyperkeratosis or eczema in the neck region, and chronic erosions of the perineal area, vulva, preputial opening, skin-horn junctions, interdigital clefts, heels and around the dewclaws.

Lameness may develop as a result of laminitis and/or interdigital necrosis. Hoof deformities may result. Anaemia, neutropenia and lymphopenia are usually present. Secondary infections are common. Affected animals ultimately succumb to progressive debilitation.

Figure 2 Cerebellar hypoplasia in a calf as a result of in utero infection with bovine viral diarrhoea virus. Compare with the normal brain. (Kindly provided by Norden Laboratories, Lincoln, Nebraska)

Pathology

Primary postnatal infections

It is difficult to differentiate between the lesions induced by severe postnatal BVDV infections and those in MD. Most reports, especially those in the earlier literature on BVDV-induced disease, dealt primarily with animals afflicted with MD as these animals had a fatal outcome. The most reliable data on primary postnatal infections are those obtained from animals that suffered or died from experimental infections.33, 87, 247

Acute postnatal BVDV infections may be subclinical, mild or severe. The basic pathological lesions are similar but vary in the degree of their manifestation in an individual animal. Some genotype 2 biotypes induce the severe lesions  indistinguishable from MD. Clinical pathological examination of the blood of acutely infected animals reveals the presence of leukopenia that is manifested as lymphopenia and/or neutropenia. In some infected animals, varying degrees of thrombocytopenia are present.

The predominant gross lesions in this form of BVD is enteritis accompanied by mucosal and submucosal oedema and petechial and ecchymotic haemorrhages especially of the distal ileum and proximal colon. Often mesenteric lymphadenitis and petechiation may develop.. Single and/or multiple oral and/or oesophageal erosions are invariably present. Enlarged haemal nodes may be evident. Some BVDV-2 viruses cause generalized petechial and ecchymotic haemorrhages in the sclera, conjunctiva, endocardium, epicardium, serosal surface and mucous membrane of the entire gastrointestinal tract, spleen, most lymph nodes, oral cavity, skeletal muscles, gall bladder, urinary bladder and testes. Osteopetrosis may be associated with natural infections.204

Microscopic lesions most severe in the alimentary, lymphoid and respiratory systems. In addition to the inflammatory lesions, the submucosal and mucosal oedema of the gastrointestinal tract is manifested by dilation of the submucosal lymphatics and separation of smooth muscle fibres of the inner layer of the tunica muscularis. Follicular lymphocytolysis in intestinal submucosal lymphoid nodules and mesenteric lymph nodes is common and lymphoid depletion of spleen parenchyma may occur. Petechial haemorrhages when present occur in the follicular and paracortical areas. The tracheitis is characterized by coagulative necrosis of mucosal epithelial cells and mucosal and submucosal neutrophilic infiltrates may be present. Infections with genotype 2 isolates have resulted also in multifocal bronchopneumonia, red bone marrow atrophy and necrosis and erosions of the mucous membranes of the gastrointestinal tract.

Prenatal (in utero) infections

Bovine viral diarrhoea virus infection of the first trimester embryo or foetus may result in death culminating in resorption, mummification or abortion. Cytopathic strains of virus are lethal for the foetus, but non-cytopathic strains may generate a persistently infected foetus.  Lesions in aborted foetuses are not characteristic and are rarely seen because of foetal autolysis. When they can be visualized, however, they consist of necrotizing lesions involving a variety of tissues.

Macroscopic lesions include enlargement of the spleen and lymph nodes and enlargement, nodularity and mottling of the liver.

Microscopic changes characterized by mononuclear infiltration, especially macrophages, may be seen in the hepatic portal areas, myocardium, spleen and lymph nodes. Teratogenic effects induced by the virus are listed under Clinical signs.

Histological examination indicates that the lesions in the central nervous system develop as a consequence of arrested development, degenerative changes and/or inflammatory processes. Cerebellar hypoplasia is initiated by vasculitis and folial oedema and is the result of the reduction of the molecular layer and granular cells and the reduction and displacement of Purkinje cells. Mononuclear infiltration and demyelination may be prominent.

Chorioretinopathy is usually the result of depigmentation and the loss of neurons and cone and rod cells. Capsular cataracts may occur in the lens associated with degeneration of the lens fibres. Mononuclear infiltration of the cornea and gliosis in the optic nerve are not uncommon. A persistent pupillary membrane may be evident.

Persistent infections

Some animals with persistent infections may show growth retardation (see Clinical signs) or may appear normal and have no macroscopic lesions. However, histopathological examination of persistently infected animals may reveal focal, interstitial nephritis with mononuclear infiltration and localized thickening of glomerular basement membranes. Encephalitis marked by mononuclear cell infiltrations, hepatitis characterized by mild mononuclear cell infiltrations in portal triads, focal peribronchiolar lymphoid hyperplasia, and focal epithelial necrosis with mononuclear cell infiltration in the tongue and the oesophagus may also occur.65, 103 The ovaries of persistently infected cows may show a significant decrease in the number of tertiary follicles, Graafian follicles, atretic follicles and corpora haemorrhagicum/luteum/ albicans.103

Mucosal disease

Mucosal disease is characterized by necrotic lesions of variable severity, primarily in the gastrointestinal tract, lymphoid organs and skin (see Clinical signs). The basic pathological changes of acute and chronic MD are generally similar but debilitation, emaciation and secondary infections are more commonly associated with the latter.

Macroscopic lesions of acute MD usually include erosions/ ulcerations in the mucous membranes/epithelia of the muzzle, lips, oral cavity, external nares, nasal cavity, oesophagus, rumenal pillars, reticulum and abomasum (Figure 3 to Figure 6). The tongue is not always affected but when it is, lesions may be present on all its surfaces. Oesophageal erosions tend to be elongated and those in the nasal mucosa and external nares are generally small. Oedema and haemorrhage may be associated with the erosions/ ulcers in the abomasum. Intestinal lesions vary in nature and extent and may appear as catarrhal, haemorrhagic, fibrino-necrotic and/or erosive/ulcerative enteritis (Figure 7). Lymphoid tissue in Peyer’s patches of the small intestine and proximal colon are necrotic and haemorrhagic, and the overlaying mucosa invariably manifests a severe inflammatory reaction. Other lymphoid tissue abnormalities include atrophy of the thymus and enlarged, oedematous peripheral lymph nodes.

Gross lesions of the upper gastrointestinal tract are often insignificant or absent in chronic MD. Skin lesions are, however, a consistent feature of chronic disease. They present as erosions and/or eczema or hyperkeratosis and are most prominent in the perineal, neck and shoulder regions, preputial opening, interdigital skin and coronary bands. Secondary bacterial infection of these lesions may occur. Eczematous lesions are characterized by hyperkeratosis, parakeratosis, hypothrichosis and/or alopecia.

Bronchopneumonia and fibrinous pleuritis due to secondary bacterial infection are present in some cases (Figure 8). Conjunctivitis also is a common feature of MD.

Histopathological lesions associated with MD include mononuclear cell infiltration and necrosis of keratinized epithelia, crypt epithelium of intestinal villi and lymphoid tissues.17, 135 The virus induces apoptosis in some cells, especially lymphocytes.

Focal mononuclear infiltration is an early event in affected keratinized epithelia usually involving the lamina propria and dermis. This is followed by haemorrhage and widespread cellular necrosis resulting in well-circumscribed necrotic foci in the stratum spinosum covered by an intact stratum corneum. Erosions develop when the integrity of the latter is lost. Hyperkeratosis and parakeratosis may be prominent in some lesions, resulting in the formation of thick keratin-like masses.

Figure 3 Ulcers of the hard and soft palate from a calf with bovine viral diarrhoea virus infection. (Reproduced by courtesy of Dr C.S. Patton, University of Tennessee)

Figure 4 Ulcers of the oesophagus from a calf with bovine viral diarrhoea virus infection. (Reproduced by courtesy of Dr C.S. Patton, University of Tennessee)

Figure 5 Ulcers of the rumen from a cow with bovine viral diarrhoea virus infection. (Courtesy of Norden Laboratories, Lincoln, Nebraska)

Figure 6 Lesions of the abomasum from a calf with bovine viral diarrhoea virus infection. (Reproduced by courtesy of Dr C.S. Patton, University of Tennessee)

Intestinal lesions are present primarily in the fundi of the crypts. Degeneration and necrosis of crypt epithelium cells result in the accumulation of cell debris and mucus in dilatated crypt lumens. Eventually there is marked atrophy of villi covered by flat epithelial cells and the lamina propria appears fibrotic and almost acellular. In some areas, squamous metaplasia of the epithelial cells may be the primary crypt lesion accompanied by marked macrophage and other mononuclear cell infiltration in the lamina propria. Oedema, hyperaemia and diapedesis may be present in the lamina propria.

Marked histopathological changes are usually present in the lymphoid tissues. In acute MD, apoptosis of thymocytes in the cortex of the thymus may be striking, and depletion of thymocytes may occur in the medulla but the architecture of the organ is retained. The presence of numerous large epithelial cells and large, active macrophages in the cortex may be a prominent feature. In chronic cases of MD, there is progressive structural disintegration of the zonal organization of the thymus and severe atrophy of the cortex.

Figure 7 Extensive ulcer of the small intestine from a calf with bovine viral diarrhoea virus infection. (Kindly provided by Norden Laboratories, Lincoln, Nebraska)

Severe depletion of mononuclear cells and architectural distortion in Peyer’s patches occur in acute MD. Chronic cases of the disease are characterized by marked atrophy of the cellular elements of the Peyer’s patches. In these cases, only hyalinized fibrotic tissue containing small foci of lymphoid tissue can be recognized. Changes in lymph nodes vary and include depletion of lymphocyte in the peripheral subsinus zones and primary and secondary follicles. Variable lymphocyte depletion may be present in the paracortex, whereas the medulla may be relatively normal or slightly enlarged. The latter is partly due to accumulation of mononuclear cells. The bronchial and retropharyngeal lymph nodes may have inflammatory reactions that reflect secondary bacterial infections in addition to mucosal disease-specific changes. In the spleen, lymphoid depletion occurs around the periarteriolar lymphoid sheath and follicular changes are similar to those in the lymph nodes. Tonsillar changes resemble those in the Peyer’s patches and the lymph nodes.

Respiratory tract disease

Bovine viral diarrhoea-associated disease of the respiratory tract depends on the presence and effect of opportunistic pathogens.178 Typically, severe fibrinous pleuropneumonia develops in animals suffering from combined M. haemolytica and BVDV infections179, 182 (Figure 8). Primary uncomplicated respiratory tract disease may include interstitial mononuclear pneumonia, alveolar epithelial necrosis and haemorrhage.147

Figure 8 Interstitial and fibrinous bronchopneumonia in a calf experimentally infected with bovine viral diarrhoea virus and Mannheimia (Pasteurella) haemolytica

Immunology

Immune response to natural infections

Responses to viral proteins

It is probable that an immune response develops to most, if not all, of the structural and non-structural BVDV proteins after infection.22, 25, 78 The virus induces both B-cell and T-cell responses.22, 132, 172, 173 Specific lymphocyte depletion studies have implicated a major role for CD4+ in controlling acute infections, but not for CD8+ cells, which may indicate MHC class 2 restricted cytotoxic T-cell activity.116 The precise immunogenic potential of specific viral proteins is not known and  immunization with live or inactivated virus elicits antibodies to numerous viral proteins.22, 31, 32 A temporal variation in the response to individual proteins has been noted; E2 and NS3/NS2-3 may be immunodominant as it relates to an antibody response as noted in western blot assays.

The major pestivirus envelope glycoprotein (E2) is the main target of neutralizing antibodies as shown with monoclonal antibodies specific for E2.72, 73 The equivalent protein in classical swine fever or hog cholera virus elicits a protective response in pigs when delivered by a live virus vector.204 Three virus neutralizing antigenic domains have been identified73 as well as nine antigenic sites on E2 of BVDV - 2.59 In contrast, epitopes susceptible to neutralization on E2 of BVDV- 1 may be clustered within a single antigenic domain.73 Epitope mapping of E2 indicated the existence of discontinuous regions of the proteins in some of the epitopes, suggesting that appropriate conformation may be necessary for an effective antibody response.165 Several of these neutralization responsive epitopes are conserved among ruminant pestivirus strains but differ from those of classical swine fever viruses.71, 164, 246 However, some BVDV E2 epitopes are not as well conserved;71, 82 four groups of BVDV isolates were defined with nine E2-specific monoclonal antibodies by some investigators,30 whereas six groups were defined by others.69, 238

The other BVDV glycoproteins, Erns and E1, do not elicit antibodies that efficiently neutralize the virus.37 Low titre neutralization activity is mediated by the BVDV equivalent of E1 in classical swine fever virus, but the neutralization mechanism seems to differ from that mediated by E2.243 Natural infections, live virus vaccines, and inactivated virus induce antibodies in calves to Erns and E1, detectable by immunoprecipitation, but these antibodies may not be important components of humoral defence mechanisms. However, the analogous proteins of classical swine fever virus, delivered in a live virus vector, induce protective immunity in pigs against challenge-inoculation with the virulent classical swine fever virus.204 E1 epitopes may be relatively conserved among BVDV strains and antibody responses to this protein correlate with neutralization antibody responses to E2.128

Although all pestiviruses have some shared antigens,68, 212 considerable antigenic variation exists among isolates of BVDV as suggested by cross-neutralization studies with polyclonal or monoclonal antibodies (see Aetiology).10, 25, 52, 53, 69, 245, 246  Multiple genotypes (subtypes) of the virus exist but there is evidence that suggests that heterotypic neutralizing antibody responses are elicited by inactivated and live isolates in calves.11, 23, 31, 32, 143 Inactivated virus, however, does not seem to induce complete protection against heterologous virus isolates in spite of these heterotypic antibodies.22, 23, 68, 143, 172 Data from some studies suggest that calves immunized with vaccines derived from BVDV- 1 isolates are protected against challenge to BVDV-2.61, 63, 64, 160, 237. Furthermore, although a clear antigenic distinction exists between the ruminant pestiviruses and classical swine fever viruses especially among the glycoprotein epitopes, data from several studies indicate that at least some ruminant pestiviruses may induce protective immunity to classical swine fever in pigs.134 This suggests that broad cross- protection is elicited by at least some pestivirus strains and begs the question on the relative importance of humoral and cell-mediated immunity to BVDV.

Few investigators have compared the relative significance of B-cell and T-cell responses with immunity to BVDV.116, 123, 131, 132 Similarly, few studies have been conducted on the development and measurement of cell-mediated immunity to BVDV.116, 172 Live virus may be necessary for the induction of cell-mediated immune response to BVDV.123, 143, 172 Immunization with inactivated virus vaccines may result only in a short-lived immunity with a narrow antigenic spectrum.23, 143, 199 Cell-mediated responses, therefore, may be an important immune mechanism for controlling primary BVDV infections.116, 132, 174 This conclusion is reinforced by the observation that immunity induced in pigs by certain ruminant pestiviruses against classical swine fever virus challenge-inoculation is not necessarily correlated with development of neutralizing antibodies to classical swine fever virus.66, 67 It has also been reported that a vaccinia recombinant, encoding the minor structural classical swine fever glycoproteins only, did not elicit antibodies in pigs but did induce protection against challenge-inoculation with the virus.204  Experimental evidence that lack of detectable serum antibody in calves does not necessarily reflect a lack of protection was reported recently; this indicates that immunological memory to this virus is not always reflected by the humoral component of the immune response,174, 195 and suggests that live virus vaccines (or perhaps recombinant vaccines using a vector capable of replicating in animals) may be required to induce a solid and broad-based immunity to this virus.

Immune responses of persistently infected animals

Deficiencies in a variety of immunologic functions have been identified in persistently infected animals (see Pathogenesis: Immunosuppression). Persistence of BVDV in cattle seems to be the consequence of B- and T-lymphocyte immunological tolerance. Neutralizing and non-neutralizing antibody to the persistent virus are usually absent in these animals. However, most persistently infected animals are immunocompetent because they respond immunologically, at least to some degree, to a variety of organisms and to heterotypic BVDV antigens.21 Persistently infected animals may respond with antibodies to neutralizing epitopes not shared with modified-live vaccine virus or wild type field strains.  If neutralizing antibody develop, these are  highly strain specific.21 The vigour of the immune response in persistently infected animals to various antigens may, however, be attenuated compared with that of ‘normal’ animals (see Pathogenesis: Immunosuppression).

Diagnosis

The complex pathogenesis of BVDV-induced disease and sometimes insidious nature of infections caused by this virus in cattle presents a significant challenge for the laboratory diagnostician.15, 39, 112, 205 The optimal procedures for diagnosing acute BVDV infections may be different from those used to detect persistently infected animals (see Interpretation of laboratory tests). With the identification of BVDV in a clinical sample, efforts should be made to determine whether one is dealing with an acute infection or a persistent infection as this may impact herd management decisions.

The accessibility to efficient diagnostic support is an essential component for the control of BVDV. Furthermore, the collection of appropriate diagnostic samples and the correct interpretation of laboratory results requires a sound knowledge of the complex pathogenesis of BVDV infections.

A particular challenge in the diagnosis of BVDV infections can be strain heterogeneity.52, 82, 85 It requires the careful selection of broad-spectrum reagents (e.g. antisera or virus strains used in the tests) and nucleic acid detection tests that can detect all strains of virus.41  Polyclonal or monoclonal antibodies directed to certain E2 and NS3 epitopes appear to be the most appropriate in this regard for tests employing anti- BVDV sera.53, 72 The ubiquitous presence of BVDV and BVDV antibodies in commercial bovine foetal serum (FBS) used in propagating cell cultures has led to spurious results in tests aimed at the detection of virus or antibodies. Experienced diagnosticians substitute horse serum for FBS in cell culture media or use irradiated FBS to eliminate all infectious agents.

Virus detection

Virus isolation

In spite of the development of a plethora of new diagnostic virus detection procedures, recovery of infectious virus in cell cultures remains the ‘gold standard’. Although it lacks some sensitivity, it is still a reliable and widely used method for detecting BVDV. Primary bovine foetal cell cultures, such as turbinate, skin and testes cells are commonly used  to grow the virus from diagnostic specimens. Some cell lines such as MDBK are also used in BVDV diagnostic systems. Most field strains are noncytopathic (see Aetiology) and can only be detected by indirect methods (e.g. immunofluorescence, immunoenzyme staining or RT-PCR assays) in the cultured cells after three to seven days’ incubation.38 Cytopathic strains which can be isolated from cases of MD cause cytopathic effects in cultured cells after several passages in the primary isolation step.

Bovine viral diarrhoea virus persistently infected animals with or without MD signs are walking “virus factories” and virtually any standard diagnostic specimen will yield infectious virus at any time.  This would include serum,  EDTA whole blood, mucosal surface swabs, and tissue specimens such as lymph node, kidney, heart, and small intestine.  The one precaution is with neonatal calves where colostral antibodies may interfere with isolation attempts.  In this instance, EDTA blood is the appropriate specimen.  For acute infections specimens are similar to those for persistently infected animals, but timing of specimen collection is critical.  For instance, virus can be isolated from serum but only within several days post- infection.  As an immune response unfolds, virus becomes complexed with antibody and eliminated for detection by virus isolation.  Attempts to recover BVDV from faeces often yield disappointing results and this sample type should therefore be avoided. Mononuclear cells in the buffy coat from acutely infected animals or persistently infected animals  are by far the best sample for detecting BVDV. Isolated mononuclear cells may be virus positive for 14-21 days post- acute infection and always positive in a persistently infected animal.

Antigen detection

The presence of BVDV in specimens can be determined rapidly and directly by the detection of viral antigens with various immunoassays.9, 39, 229, 230 The detection and location of the virus in tissues can be done by immunofluorescence (not good for persistently infected animals) and immunohistochemical (IHC) methods on sections of frozen tissues such as lymphoid tissues, heart, kidney, intestine and skin. The IHC tests have been adapted for use with formalin- fixed, paraffin-embedded (FFPE ) tissues.229, 230 A major advance was the identification of an Mab specific for Erns (E0) that worked for IHC testing using FFPE tissues.161  This finding permits the detection of BVDV when the primary differential testing failed to recognize the role of persistently infected animals in “non-BVDV” cases and the only material available is formalin-fixed tissues.

Several antigen-capture, enzyme-linked immunosorbent assays (ACE) have been developed for detection of BVDV in blood, leukocytes and tissue samples.38, 100, 207 These types of assays are only appropriate for detecting persistently infected animals as the tests are not sensitive enough to detect the lower amount of antigen in acutely infected animals This procedure exploits broad-spectral monoclonal (or polyclonal) antibodies attached to an appropriate surface to trap viral antigens that are then identified by a second BVDV specific antiserum tagged with an enzyme detector.  Assays based on detection of NS3 generally lacked sensitivity and required sample processing to release the antigen from infected cells.  A test based on detection of Erns (E0) in serum, tissue samples, and individual milk samples overcame these issues, showing nearly 100 per cent sensitivity for detecting persistently infected animals.206

Antibody detection (serology)

Antibodies to BVDV in serum and milk may be detected by several methods, the most common of which is quantification of antibodies capable of neutralizing virus infectivity (virus neutralization test). Paired (acute and convalescent) serum samples are required to confirm BVDV infection unless a herd analysis is done. Serological procedures are subject to test variation and therefore paired samples should be simultaneously tested to eliminate between test variations.

Virus neutralization

The major target of virus-neutralizing antibodies is the surface E2 protein, one of the most variable of the BVDV structural proteins (see Aetiology).

Therefore, the test has variable specificity for particular virus strains depending on the affinity of antibodies for particular E2 epitope(s) present in the serum.41, 166, 167 Consequently, the test is often isolate specific. To ensure broad reactivity for this assay, sera must be tested against more than one reference isolate.41 Generally, cytopathic BVDV isolates are used in virus neutralization tests as the endpoints can easily be determined microscopically.  Non-cytopathic strains can be used but virus growth must be determined indirectly by immunofluoresence or IHC.  Commonly strains for VN tests include NADL, Singer or Oregon C24V, but any isolate can be standardized for use in serological assays. . However, these traditional isolates are all BVDV-1 serotypes and with the recognition of BVDV-2 and Hobi strains, the choice of strains in neutralization assays may need to be broadened to reflect local needs.10, 11

Enzyme immunoassays and immunofluorescence

Results of some studies indicate that antibody detection ELISA tests with purified or recombinant test antigen coated onto plates correlates well with virus neutralization tests for the detection of BVDV antibodies.126, 128, 154, 188 A competitive ELISA for evaluating antibody responses to the conserved NS3 protein has been developed. Because of the requirement for extensive purification/preparation of appropriate BVDV antigens, ELISA applications have had mixed acceptance,209 but recent developments  improved the specificity and sensitivity of these assays and some are available commercially.126

Nucleic acid detection

Nucleic acid detection methods have the potential for superior specificity and sensitivity but require that viral genomic base sequences are known.112 Unlike virus isolation, the presence of neutralizing antibody in samples from live animals does not interfere with virus detection by nucleic acid assays.112 Additionally, these methods react not only with genomic RNA from infectious virions but also with RNA from inactivated or defective virus and RNA in infected cells. This adds to the relative sensitivity of the test.  An issue with interpretation of test results is that the nucleic acid detection tests can also react with vaccines, particularly modified-live vaccines.

The polymerase chain reaction amplification (RT-PCR test) is based on the capacity of synthetic DNA oligonucleotides (primers) to bind specifically to complementary genomic target sequences.84 Ribonucleic acid targets (such as the BVDV genome) are first converted to cDNA by reverse transcription before PCR amplification takes place (RT-PCR). Primer selection is critical; it may allow broad identification of all pestiviruses, discrimination among the four recognized species of this genus (BVDV-1, BVDV-2, border disease virus, and classical swine fever/hog cholera virus) or discrimination among BVDV genotypes (subtypes).84  The procedure has been adapted for various specific applications, such as detection of viral RNA in formalin-fixed, paraffin-embedded tissues and in bulk milk samples.105, 185, 191 The superior sensitivity and versatility of the PCR procedure is appealing and with the introduction of standardized reagents and protocols plus automated extraction and testing equipment, RT-PCR testing for pestiviruses is at the forefront of diagnostic testing.

Interpretation of laboratory tests

Primary postnatal infections

Cattle with acute BVDV infections are viraemic and virus may be detected from three to 21 days after infection depending on the specimen and test used. Viral RNA detection by PCR extends the window of detection well beyond that found for infectious virus especially when blood mononuclear cells are used as the specimen. Attempts at virus recovery from contact animals may improve the chances of diagnosis in a herd. Tissues from aborted foetuses may not yield a BVDV “signal” due to the fact that  the foetus may not be expelled until several weeks following the infection.  In some instances, these foetuses may have BVDV antibody which may have diagnostic significance. However, because of its ubiquity, the presence of BVDV in diseased animals or in an aborted foetus should not automatically imply a primary aetiological relationship. A complete history, clinical signs, a careful consideration of test results make it imperative that the possible presence of other pathogens be taken into account. However, the detection of BVDV in a clinical sample should never be ignored as the presence of BVDV in an animal from a production herd is a serious health issue.

Persistent infections

Persistently infected animals were generally identified by sequential isolation of virus from mucosal swabs or whole blood taken at  intervals of 21-30 days. Within this timeframe, acutely infected animals should have no detectable infectious virus.  Due to the increased sensitivity of RT-PCR assays, the interval of the confirmatory test may need to be extended.  With ACE tests that  primarily detect persistently infected animals, retesting of the animal may not be necessary particularly if other characteristics of persistently infected animals are evident such as poor growth rate, dermatitis, and chronic pneumonia. In most of these animals the level of viraemia is high (up to 106 infectious doses per millilitre) but it may be lower in some (102 infectious doses per millilitre). The level of viraemia may decrease gradually in older animals. Colostral antibodies may interfere with virus detection in animals younger than three months. Therefore, washed viable leukocytes should be used in attempts at viral isolation from animals of this age group.  Antigen capture ELISA (ACE) testing on skin biopsies is generally not affected by colostral antibodies, but testing animals several weeks old is recommended.  RT-PCR tests are also not affected by colostral antibodies.

Although persistently infected animals are immunotolerant, they may develop antibodies to heterologous isolates of the virus including modified-live vaccines Consequently, the practice of attempting to identify persistently infected animals by first screening a herd for antibody-negative animals is unwise. Persistently infected animals should be identified by testing all animals in a herd for viraemia or antigen positive skin biopsies. Pooled-sample testing which reduces the cost of herd screening is feasible particularly when using RT-PCR assays.81, 151 Serum samples from adult animals and unclotted whole blood from calves under three months of age  can be used for this purpose. Additionally, all animals born into the herd  following seven months after the removal of the last persistently infected animals should also be tested for viraemia to insure that a persistently infected foetus did not develop while any BVDV was in the herd.

Differential diagnoses

Bovine viral diarrhoea disease must be differentiated from conditions in cattle causing diarrhoea, erosions/ulcerations of the gastrointestinal tract, reproductive failure, teratology, skin disease, laminitis, poor growth and respiratory tract disease. The causes of these conditions are very broad and include many diverse infectious agents, parasites and toxins. The most common of these are malignant catarrhal fever, bluetongue, epizootic haemorrhagic disease of deer, infectious bovine rhinotracheitis, salmonellosis, foot-and-mouth disease,  cryptosporidiosis, coccidiosis and helminthosis.

The prevalence and epidemiology of these diseases in different locations must be taken into account in developing an initial diagnostic list. Foot-and mouth disease is prevalent in several countries but it is generally characterized by low mortality, absence of diarrhoea and an explosive spread. Malignant catarrhal fever is usually associated with the presence of certain wild ungulates i.e. wildebeest (Connochaetes spp.) or sheep and has a very high mortality.

The mortality and morbidity rates of BVDV infections are influenced by many factors including virus strain, herd immunity, stage of gestation (for foetal disease), the occurrence of opportunistic infections and the nature of the BVDV infection (primary or persistent).

Control

Advances in the understanding of the epidemiology, pathogenesis and immunity of BVD have enabled development of rational strategies for controlling disease syndromes caused by the virus.19, 23, 199 The most appropriate control programme is determined by the nature of the animal population (age, pregnancy status), production system, stress levels, government regulations and BVDV-infection history. The general guidelines for controlling BVDV in a herd should be based primarily on eliminating the source of virus (e.g. persistently infected animals) and secondarily with breaking the infection cycle (i.e. vaccination).23, 199 Non-vaccinated herds without persistently infected animals are particularly at risk of suffering severe losses if the virus is introduced. In these instances, biosecurity standard operating procedures must be developed and actually implemented.

The primary goal of a control strategy is the removal of persistently infected animals from a herd and prevention of foetal exposures.19  Since the only way a persistently infected animal can be produced is through infection of the foetus, an animal need only be tested once in its lifetime for persistently infected status. All cattle should be initially screened for the virus, including all calves born into the herd from cattle pregnant at the initiation of the testing program. Animals introduced into the herd should be tested prior to their introduction. If an animal is to be introduced into a non-vaccinated herd, it is recommended that it be quarantined during the testing of two consecutive samples taken approximately 30 days apart. Calves from introduced animals should be tested for virus immediately after birth before they have made contact with susceptible animals.

Semen and embryos used in the herd should also be free of virus. Donors of these products and media used for their dilution or transport should be free of the virus. Precautions to prevent fomite transmission of BVDV from adjacent premises must be implemented. Too frequently, contact with neighbouring ruminants introduces BVDV into a negative herd. Assessment of these contact risks should be part of the biosecurity plan for the production unit.

The most effective mechanism for breaking the cycle of BVDV infection between persistently infected animals and other animals is to ensure that the latter are immune. This should prevent foetal infections and thus the opportunity for generating new persistently infected animals. Generally, immunity in the dam prevents transplacental infection. Therefore, a rational approach to vaccination, in addition to reducing the sources of infection, may be the most effective strategy for controlling BVD with minimal risk.23 It has been estimated that a vaccine coverage of approximately 60 per cent is required to prevent episodes of disease in herds without persistently infected animals and that an almost 100 per cent coverage is necessary when persistently infected animals are present.57, 113

The first BVDV vaccines were produced from modified live virus157. Controversy concerning the safety of this vaccine has existed almost from its inception. Problems associated with its use that have been reported include induction of MD, impaired immunity to other agents, and foetal disease.18, 23, 137, 198 Some early reports of vaccine failures did not fully appreciate the existence of persistently infected animals and their impact on diseased following vaccination. In addition, early vaccines may have been a mixture of  BVDV strains coming from contaminated cells or sera  which cast aspersions on the safety of the vaccine strains.

There is strong evidence that indicates that live virus vaccines induce a more durable immunity than killed vaccines.64, 177 With the widely accepted view that prevention of foetal infection is the key to BVDV control, multiple studies have been done to assess foetal protection particularly with the use of modified live vaccines.  The data are unequivocal that foetal protection can be achieved with vaccination, but strain variation of field isolates may not permit 100 per cent protection in all cases.159 To be clear, there are no universal BVDV vaccines and local strains of virus should be assessed as to their similarity to the available vaccines.

Live virus vaccines induce antibody responses to a variety of structural and non-structural viral proteins. The neutralizing antibody response is rather narrow at first, but by three weeks post-inoculation, a broader neutralizing response may develop.31 The immune response to BVDV infections includes cell-mediated responses (CMI)  in addition to the neutralizing antibody response.132, 143, 173 The relative importance of the CMI responses as compared to antibody responses is not known, but they are major contributors to the immune status of an animal to the virus. They may be the critical factor responsible for the superior immunity induced by live virus vaccines as compared to that induced by inactivated viruses. The latter do not elicit adequate T-cell responses.172 Little information is available on how long live virus-induced immunity persists but experimental evidence suggests, based on neutralizing antibody determinations, that it persists for at least 18 months.64 The most valid evaluation of a vaccine is its capacity to induce foetal protection in pregnant animals and modified live vaccines have withstood this scrutiny best.62, 159, 160

Evidence suggests that recombinant vaccines such as vaccinia virus incorporating genomic sequences of pestivirus immunogens may have an advantage over live virus BVDV vaccines by eliminating the potential  deleterious effects on the foetus and immunosuppression.204, 239 A new double mutant modified live vaccine appears unable to initiate foetal infections which removes a concern of use of modified-live vaccines with pregnant animals.148

Inactivated BVDV vaccines are undoubtedly safe, but their efficacy against heterologous virus challenge is variable perhaps due to a lack of a CMI response. Foetal protection has been demonstrated in animals inoculated with inactivated vaccines.45 The antibody response to E2, present on the surface of the virus, is immunodominant.22, 23, 25 Experimental subunit vaccines based on the E2 glycoprotein are also promising.34, 43, 49, 109 Without evidence for a strong CMI response induced by inactivated vaccines, neutralizing antibodies are paramount for protection.  That being the case, it is essential for inactivated vaccines to contain the antigens known to be prevalent for the BVDV strains circulating in a given region.

A common problem with the use of inactivated vaccines is the failure of farmers to administer the required secondary and booster inoculations after primary vaccination.187 This may be an inherent drawback to including a killed virus component in a multivalent vaccine in which the other components are primarily modified live viruses.

It may be concluded that an effective broadly cross-reactive immunity to BVDV cannot be achieved with inactivated whole viruses or with subunit products of the virus. Live virus vaccines are effective immunogens, but safe use of the products must be built into a herd management program.   Recombinant antigens vectored in live viruses, such as has been done with classical swine fever virus, deserve serious consideration for the control of BVD.137, 204

Several general guidelines for vaccination should be considered.23 Calves under three months of age are not generally vaccinated because of potential vaccination failure associated with the presence  of colostrum- derived antibodies. Calves should therefore be vaccinated at the age of four to six months. Calves that do not receive colostrum (e.g. veal calves) should be vaccinated soon after birth. Heifers and cows must be revaccinated 30 to 60 days before breeding to reduce the chances of foetal infections. Standard modified live virus vaccines should not be used in pregnant animals nor in stressed populations.

Vaccination is but one component of any effective BVDV control program. The elimination of all persistently infected animals in a herd is the necessary and critical first step to control.  This should then be followed by a tailored vaccination program, along with the implementation of stringent biosecurity measures. Finally, an ongoing surveillance program should be implemented to detect any introduction of BVDV into the herd.

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