CHAPTER 50 Viral Diseases of the Fetus
Bovine viral diarrhea virus (BVDV) is one of the most commonly encountered and economically important pathogens of cattle in North America. Since the mid-20th century, BVDV has been recognized as a significant cause of disease of the gastrointestinal system. The impact of BVDV on reproduction was not perceived for another 30 years, when the occurrence of persistent infection in immunotolerant cattle was described.
BVDV infections may occur in cattle as acute illness—that is, bovine viral diarrhea (BVD)—or as a generally chronic condition—mucosal disease. When susceptible pregnant cattle are infected with BVDV, transplacental infections usually occur. Transplacental infections may lead to embryonic or fetal death and abortion, to developmental defects of organs, or to development of immunotolerance and establishment of persistent infections. Acute BVDV infections contribute, through immunosuppression, to causing multifactorial diseases, such as diseases of the respiratory and enteric tracts in susceptible calves.
The clinical form of BVDV infection—inapparent or severe BVD, reproductive failure, persistent infection, or mucosal disease—observed within a herd is dependent on interaction of several factors at the time of infection. These determining factors include the biologic properties of the virus, the age and stage of gestation of pregnant cattle, level of immunity of the herd, and the interplay of stressors.1
BVD is an acute postnatal infection in seronegative, immunocompetent cattle. The clinical severity of acute BVDV infections is variable, but a majority of postnatal BVDV infections are inapparent. Milder forms of BVD are characterized by high morbidity, low mortality, a normal host immune response, and minimal mucosal lesions. Usual findings include pyrexia, nasal discharge, and transient leukopenia. Viremia lasts for 3 to 10 days (acute infections with higher virulence isolates may result in viremia of longer duration) and antibody titers rise slowly for 3 months after infection.2 Severe acute BVD outbreaks with marked thrombocytopenia, hemorrhages, and high mortality rates have been associated with infection with high-virulence BVDV isolates.
Acute BVDV infections contribute to causing multifactorial diseases through immunosuppression. Immunosuppression is mediated by suppression of immune functions through the lymphotropism of BVDV. BVDV lymphotropism results in depletion of lymphocytes from lymphoid tissues. Immunosuppression due to BVDV infection enhances the severity of bovine rotaviral enteritis in calves, in addition to directly causing enteritis.3 BVDV-induced immunosuppression predisposes calves to development of naturally occurring bovine respiratory tract disease (BRD). Indirect effects of BVDV in causing BRD were demonstrated in experimental bovine respiratory syncytial virus (BRSV) and BVDV co-infections in which more severe respiratory tract and enteric disease occurred than in infections with either virus alone.4 Subpopulations of lymphocytes were more markedly altered in peripheral blood and lymphoid tissues from co-infected calves than in calves infected with either BRSV or BVDV alone. Co-infected calves had a reduction in the percentage of T lymphocytes (including CD8+ lymphocytes and CD4+ lymphocytes) in the thymus and Peyer’s patches.5 An additional finding in these calves was more extensive pneumonia, characterized by caudodorsal as well as cranioventral interlobular edema, emphysema, and bronchopneumonia in caudal lung lobes. By contrast, calves infected with BRSV alone had only cranioventral bronchopneumonia.
Transplacental infection is likely to occur in susceptible, pregnant cattle infected with BVDV. The outcome of transplacental infection is dependent on the biologic properties of the infecting virus, especially the biotype of the virus, and the stage of gestation at the time of infection. The potential outcomes of transplacental infections—embryonic or fetal death and abortion, developmental defects of organs, and development of immunotolerance with establishment of persistent infections—are discussed later in the chapter.
Fetal infection with noncytopathic BVDV can result in the birth of calves with persistent BVDV infection. The primary means of producing a persistently infected calf is through transplacental infection after a primary acute infection in a pregnant cow, although persistently infected cows (i.e., congenitally infected) also will give birth to persistently infected calves. Persistently infected animals shed large amounts of virus and are therefore carriers and a primary source of exposure for susceptible cattle.6 In most instances they do not produce detectable antibodies to BVDV, because they are immunotolerant to the virus. Some calves with persistent infection are stunted or weak at birth, have poor growth rates, and die at a young age.7 Others appear healthy and survive to maturity. The prevalence of cattle with persistent infection is variable; however, on the basis of sampling of randomly selected herds, it has been estimated that 4% of herds in the United States have persistently infected calves.8 Such animals are at risk of developing mucosal disease.
Mucosal disease, associated with high mortality rates, occurs sporadically (low morbidity) in cattle that usually are between 6 months and 2 years old but may be of any age. Characteristic clinical manifestations include anorexia, pyrexia, diarrhea, loss of condition, and death.2 Gross pathologic lesions may include erosive or ulcerative lesions on the muzzle and lips, buccal mucosa, and tongue. Commonly, elongated ulcerative lesions occur in the mucosa of the esophagus. Erosions also may be found on the rumen pillars, reticulum, and abomasum. Enteritis may be evident and may vary in presentation from catarrhal to hemorrhagic to erosive/ulcerative. Peyer’s patches and lymphoid tissue in the proximal colon may be hemorrhagic.9 Thymus atrophy and enlarged peripheral lymph nodes are prominent features.
BVDV is a member of the genus Pestivirus, family Flaviviridae,10 which also includes border disease virus of sheep and hog cholera virus. Pestiviruses are small, enveloped, single-stranded, positive-sense RNA viruses that are antigenically related.
The host range for BVDV comprises domestic or wild ruminants and swine. Pestiviruses are presumed to persist in the environment for no more than two weeks, and are readily inactivated by common disinfectants. Therefore, virus transmission is primarily vertical or by inhalation or ingestion of material contaminated with infected body secretions and excretions (saliva, oculonasal discharge, urine, feces, semen, uterine secretions, placenta, and amniotic fluid) of infected animals.
Isolates of BVDV vary in their relative virulence potentials, which accounts in part for variability in severity of lesions and clinical disease among different cases.11 BVDV isolates are divided into two biotypes (groups of viruses with the same genetic composition) based on their ability to induce microscopically visible changes (vacuolization and lysis) in host cells in vitro: cytopathic and noncytopathic. BVDV strains are divided into two genetic groups or genotypes—1 and 2—using gene sequencing techniques and cross-neutralization assays. RNA viruses, including BVDV, are prone to mutate; therefore, BVDV has high potential to mutate in response to selective immune pressure. Mutation is the putative strategy used by BVDV to escape the host’s immune response and to persist in the cattle population. Antigenic diversity among field isolates has important implications for development of protective immunity.
The two biotypes of BVDV, cytopathic and noncytopathic, have separate biologic roles2; biotype differences are important in disease pathogenesis. Both biotypes of BVDV infect cattle and cause disease, but only the noncytopathic isolates cause persistent infections. Isolates that have the ability to cause microscopically visible changes in host cells (vacuolization and lysis) are assigned to the cytopathic biotype. Isolates lacking this capability are assigned to the noncytopathic biotype. Cells infected with cytopathic BVDV have an 80-kilodalton (kD) polypeptide that is distinguishable electrophoretically from cells infected with noncytopathic viruses, which do not have the polypeptide.12 This 80-kD nonstructural viral protein apparently plays a crucial role in replication of cytopathic viruses. Diversity in antigenicity among strains is not discretely separable. No link exists between biotype and antigenicity, and strains that are antigenically distinct overlap both biotypes, so protective immunity afforded by a vaccine is not dependent on the biotype of the vaccine virus.
In the laboratory, the presence of noncytopathic BVDV constitutes a significant quality control issue for workers in diagnostic laboratories as well as for manufacturers of vaccines. This is because noncytopathic BVDV isolates commonly occur in commercial fetal calf sera used to supplement cell culture media used in cell cultures to grow viruses. In the diagnostic laboratory, when noncytopathic BVDV occurs undetected as a contaminant of cell culture, accuracy of diagnostic laboratory assays, such as virus isolation tests and serum neutralization tests, is compromised. Noncytopathic BVDV contamination of modified live virus vaccines during the manufacturing process has represented a significant risk factor since these products were introduced.13 The potential for contamination of cell cultures with noncytopathic BVDV is a continual concern, because between 20% and 50% of commercial fetal bovine serum lots are virus positive14 for both genotypes.15 Fetal bovine serum quality assurance procedures applied before use in diagnostic laboratory testing or in cell culture production systems to grow vaccine virus include rigorous virus testing followed by the additional precautionary measure of irradiation or chemical treatment.14
BVDV strains are divided into two genetic groups or genotypes using gene sequencing techniques and cross-neutralization assays.16,17 Genotype 1 isolates are primarily classic laboratory reference and vaccine strains. Genotype 2 viruses are found predominantly in fetal bovine serum, persistently infected calves born to dams vaccinated against BVDV, and the more recently described BVDV strains associated with high mortality and acute and peracute infections involving hemorrhage. Biotype, genotype, and antigenic cross-reactivity vary independently,18 as do biotype, genotype, and pathogenicity.19 The antigenic differences between genotype 1 and genotype 2 isolates and the clinical importance of genotype 2 BVDV isolates constitute the basis for the recognition that to be effective, vaccines must provide broad cross-protective immunity against both genotype 1 and 2 isolates.
BVDV enters the susceptible host primarily by the oronasal route and replicates in tonsils, lymphoid tissues, and epithelium of the oropharynx. Phagocytic cells take up BVDV or virus-infected cells, or both, for transport to lymphoid tissues.20 Viremia is evident 2 to 4 days after exposure. Viremia in a pregnant female is certain to lead to transplacental infection and fetal infection. The outcome of fetal infections with BVDV is determined primarily by the stage of fetal developmental at the time of infection, and by biotype and virulence of the infecting virus.21 The stage of development of the evolving fetal immune system at the time of infection plays a major role in determining the outcome of infections.21 Transplacental infections are particularly damaging during the first two trimesters of gestation and may result in persistent infections, fetal death and abortion, or congenital developmental defects.22 Persistent infection in calves is the most significant outcome of fetal infection because of the negative effects such infection has on herd production. Persistently infected calves are the most important source of virus to perpetuate disease within and between herds. Moreover, persistently infected calves usually have poor growth rates and die at a young age. Reproductive failure mediated by abortion and birth of calves with congenital abnormalities also are significant outcomes of fetal BVDV infections that adversely affect herd performance.
Persistent BVDV infections may be established if infection of the fetus occurs during the third or fourth month of gestation before immunocompetence becomes established.21,23 Viremia of the pregnant dam, stemming from either a persistent or an acute infection, is the source of the virus that infects the fetus. Before infecting the fetus, BVDV replicates in the placenta. Persistent viremia develops as a result of fetal immunotolerance and failure to develop antibodies against the persisting virus.6 Persistently infected calves are carriers because they are viremic and shed virus continuously, and they may spread virus within and between herds. The level of viremia may decline with the development of neutralizing antibody and become undetectable as the animal ages24 as a result of deterioration of highly specific immunotolerance to the persisting virus.25 Deterioration of immunotolerance, eventuating in an immune response, may result from development of antigenic-variant viruses within the immunotolerant, persistently infected animal.25 Persistently infected calves frequently are “poor doers,” have reduced growth rates, are more susceptible to common calfhood infections of mucosal surfaces including pneumonia and enteritis, and are at risk of developing mucosal disease.26,27
BVDV infections of fetuses during the first and second trimesters may cause fetal death and abortion. Third-trimester abortions also have been attributed to BVDV infection.21 Individual isolates probably vary in their ability to cause abortion. The rate of abortion under field conditions is variable, but abortion rates as high as 40% have been reported after experimental infections on day 100 of gestation.22
Congenital defects may result if infection occurs during midgestation (100–150 days). Congenital defects associated with BVDV infections may involve the nervous system (microencephaly, cerebellar hypoplasia, hydranencephaly, hydrocephalus, and hypomyelination), eye (cataracts, retinal degeneration, optic neuritis, and microphthalmia), immune system (thymic aplasia), integumentary system (alopecia and hypotrichosis), musculoskeletal system (brachygnathism, growth retardation, and arthrogryposis), or respiratory system (pulmonary hypoplasia).28 The pathogenetic mechanisms for development of defects are not known. Because fetal organs and immune system (inflammatory response) are developing during this stage, direct cell damage by viral infection and destruction of virus-infected cells by the evolving immune system are possible mechanisms.21
The outcome of BVDV infections during late gestation (last trimester) is comparable with that with acute postnatal infections of cattle. At this time the fetal immune system has developed to respond efficiently against BVDV infection. Consequently, transplacental infections during late gestation are not associated with a significant level of congenital defects. Third-trimester abortions have been attributed to BVDV infection.21 The most common outcome of infections during this period is birth of a clinically normal calf with high levels of precolostral antibodies.21,22
Persistently infected carrier cattle are identified in herds on the basis of tests conducted in a diagnostic laboratory. The tests include (1) the virus isolation (VI) test, (2) the immunohistochemistry (IHC) test, (3) the polymerase chain reaction (PCR) assay, and (4) the enzyme-linked immunosorbent assay (ELISA).
The standard VI test format (macrotest), a highly reliable test,7,29 is not practical for testing a large herd. The standard VI test may be used to test mononuclear cell preparations (buffy coats) from blood samples collected in tubes with anticoagulants. The cells are washed to limit interference from antibodies, which reduce test sensitivity. An adaptation of the standard VI test is the immunoperoxidase microtiter plate VI assay, which is relatively sensitive and specific and is designed to efficiently test large numbers of serum samples, such as in herd testing programs.30,31 Blood is collected for virus isolation from calves that are 2 months of age or older, when maternal antibody titers have declined, because maternal antibodies reduce the ability to isolate BVDV from the serum of younger persistently infected cattle.7,29
The IHC test is conducted on skin biopsy specimens (ear notches) collected from animals of any age, fixed in formalin, and submitted to the diagnostic laboratory.32–34 The fixed skin specimens are sectioned, stained, and examined for the presence of BVDV antigen. The IHC test, like the VI test, has excellent sensitivity and specificity.34 Sensitivity of IHC studies is not affected by the presence of maternal antibody, so calves of any age, including newborn calves, may be tested.34,35
The PCR assay may be used to test individual animals (serum, whole blood, or skin samples) or to screen entire herds by testing pooled samples such as bulk tank milk or pooled serum samples for the presence of carrier cattle.36 The BVDV PCR assay is highly sensitive, but a potential complication with the assay is lack of test specificity, so that false positive results are possible from nonspecific reactions with contaminating viral RNA (unpublished observation). It is therefore advisable to confirm positive BVDV PCR assay results with VI tests.35
The ELISA may be used to test individual blood samples for the presence of BVDV antigen. The antigen-capture ELISA compares closely with virus isolation techniques for detection of persistently infected cattle using blood samples routinely submitted for BVD diagnosis. The test format is adapted to a microtiter plate assay, which permits efficient testing of large numbers of serum samples.31,37
Diagnosis of BVDV as the cause of abortion is not unequivocal, because fetal infection may not result in abortion. Therefore, the presence of virus, viral antigen, or BVDV antibody in an aborted fetus does not confirm that BVDV was the cause of abortion.28 The entire fetus should be submitted to a diagnostic laboratory for complete testing because of the complexity of the factors to be considered in conclusively establishing BVDV infection as the cause of abortion or, conversely, in ruling out BVDV infection as the cause of abortion. Diagnosis of BVDV as the cause of abortion is based on evidence of BVDV infection of the fetus (presence of virus, antigen, or RNA in tissues, or antibody in serum or exudates), in conjunction with clinical confirmation of microscopic lesions, most often in fetuses aborted before 4 months of gestation. Microscopic lesions attributable to fetal BVDV infection include a necrotizing inflammatory reaction with mononuclear cell infiltration in several tissues.38 Other features may include lymphoid depletion of the cortex of the thymus, precocious development of secondary lymphoid tissue, and peribronchiolar lymphonodular hyperplasia. The cerebellum is affected with necrosis and depletion of cells and infiltration of mononuclear cells. Microfocal lesions may be seen in the oral mucosa and in the skin. Skin lesions are characterized by hyperkeratosis and parakeratosis. BVDV antigen may be deposited in lymphoid tissues and in the cerebellum.9 Demonstration of rising BVDV antibody titers in paired serum samples from dams may be not be possible. This is because antibody titer may have already increased at the time of abortion because of the time lag from infection of the dam to abortion. Identification of BVDV in a fetus in the absence of lesions provides useful information regarding the temporal occurrence of BVDV within the herd.