Immunohistological detection of bovine viral diarrhoea virus antigen in the central nervous system of persistently infected cattle using monoclonal antibodies

In a total of 25 cattle persistently infected with bovine viral diarrhoea virus (BVDV) the distribution of viral antigens in the central nervous system was studied. Using a panel of monoclonal antibodies (anti pestivirus C16; anti cytophathic BVDV C38; anti cytopathic and non-cytopathic BVDV C42; anti gp53 BVDV CA-1 and CA-3) and the indirect immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus. Morphological cellular alterations were not seen. Reactive perivascular lymphocytic infiltrations were occasional findings.     

Distribution of bovine virus diarrhoea viral antigens in the central nervous system of cattle with various congenital manifestations.

Distribution of bovine viral diarrhoea virus (BVDV) antigens in the central nervous system (CNS) of 26 cattle persistently BVDV infected, 11 cattle with mucosal disease (MD), and 32 calves with congenital brain malformations was studied using monoclonal antibodies against BVDV epitopes. In persistently infected cattle and in cattle with MD, a widespread infection of neurons was present. Predilection sites for BVDV antigens were the cerebral cortex and the hippocampus. In calves with congenital encephalopathies, viral antigen-containing neurons could only be detected in the CNS of four animals. From the topographical distribution of BVDV antigens in these four postnatal cases with end-stage lesions, no conclusions could be drawn concerning the pathogenesis of BVDV-induced encephalopathies.     

Bovine viral diarrhea virus type 2-induced meningoencephalitis in a heifer

The brain from a 15-month-old, black female Angus, with a 48-hour history of central nervous system disease, was submitted to the Oklahoma Animal Disease Diagnostic Laboratory. Microscopic findings consisted of acute, multifocal meningoencephalitis, with neuronal degeneration and necrosis and gliosis. Viral isolation yielded noncytopathic bovine viral diarrhea virus (BVDV). Virus genotyping classified the virus as BVDV type 2. Immunohistochemical labeling for BVDV antigens with BVD MAb 3.12F1 clone was prominent in the cytoplasm of neurons, glial cells, ependymal epithelium, perivascular macrophages and spindle cells, smooth muscle cells, and intravascular monocytes of the cerebrum and brain stem. Laboratory results support that tissue alterations occurred as a result of BVDV type 2 infection. In the absence of other clinical signs related to BVDV infection and using the microscopic and laboratory evidence presented, we propose that the BVDV type 2 isolated from this case may represent a neurovirulent strain of the virus. To the best of our knowledge, this is the first report of brain lesions and neuronal viral antigen localization in BVDV genotype 2 viral infection, acquired either congenitally or postnatally.     

Rift Valley fever virus-induced encephalomyelitis and hepatitis in calves.

Three calves (Nos. 1, 2 = 7 days old; No. 3 = 21 days old) were inoculated subcutaneously with virulent Rift Valley fever (RVF) virus. All calves became viremic and clinically ill, but the two 7-day-old calves were moribund and were euthanatized subsequently on post-inoculation day (PID) 3. Highest viral titers were measured in the serum, with lesser concentrations in the brain, heart, spleen, and liver of these animals. Viral antigens were detected by immunohistochemical analysis only in the livers, where positive staining was localized in coalescing foci of hepatocellular necrosis. The 21-day-old calf appeared to recover after viremia and pyrexia but became lethargic and ataxic and was euthanatized on PID 9. The calf was no longer viremic, and RVF virus was isolated only from the brain. Microscopic examination of the central nervous system revealed diffuse perivascular infiltrates of lymphocytes and macrophages, multifocal meningitis, and focal areas of neuronal necrosis and aggregates of macrophages, lymphocytes, and neutrophils throughout all regions of the brain and cervical spinal cord. There was positive immunohistochemical staining for viral antigens within the cytoplasm of neurons and glial cells throughout the central nervous system. Thus, RVF virus can cause encephalomyelitis in calves, and the specific virologic diagnosis can be made by immunohistochemical localization of viral antigens in formalin-fixed tissues.     

牛病毒性腹泻病毒间接ELISA方法的建立

持续性感染和免疫耐受是牛病毒性腹泻病的重要特征,也是该病的防控难点.本试验将2种生物型的牛病毒性腹泻病毒(BVDV):致细胞病变(CP)型(BVDV OregonC24株)和非致细胞病变(NCP)型(BVDV Yak株),灭活、浓缩作为抗原,分别免疫家兔制备高免血清.同时,使用BVDV全病毒蛋白作为包被原,建立了检测BVDV的间接ELISA检测方法.本试验利用该方法对高免血清进行检测,探讨CP型BVDV与NCP型BVDV抗原免疫原性差异.结果显示,CP型BVDV的高免血清效价均比NCP型BVDV高免血清的效价高,平均高35%.结果表明,CP型BVDV的免疫原性优于NCP型BVDV,且CP型BVDV与NCP型BVDV的血清效价具有明显差异.这为在实践中疫苗毒株筛选及生产提供了理论指导.     

Pestivirus in cattle: experimentally induced persistent infection in calves

Twenty-two heifers were infected intranasally with non-cytopathic bovine viral diarrhoea virus (BVDV) between days 74 and 82 of pregnancy. All animals had developed serum antibodies against BVDV 5 weeks later. No clinical effects were seen in the heifers, and they all delivered a live calf. The newborn calves were generally small, appeared unthrifty as typical 'poor doers', and some developed secondary infections with diarrhoea and signs of respiratory disease. Eighteen of the 22 calves were born without antibodies against BVDV and were persistently infected (PI) with the virus. One was weak at birth and died the following day. Four calves were born with serum antibodies against BVDV and with no detectable virus. Three of these showed signs and/or pathological changes indicating disease in the central nervous system. Otherwise, there were no obvious clinical differences between these calves and the PI calves, nor were there any apparent significant differences in blood parameters between these groups. In general, the calves showed low gamma-globulin values and thrombocytopaenia, but moderately increased fibrinogen values and relatively normal lymphocyte numbers.     

Modulation of the cellular immune responses to T-cell-dependent and T cell-independent antigens in lambs with induced bovine viral diarrhea virus infection

Functional interaction between lymphoid cells and lymphotropic viruses is particularly evident for bovine viral diarrhea virus (BVDV) in cattle and its closely related virus, the border disease virus (BVDV) in sheep. The most important aspect of acute or chronic phases of BVDV or BDV infection was the host's increased susceptibility to secondary bacterial or viral infection. To study the ability of BVDV to alter the development of the cellular immune responses to concomitant inoculation with T cell-dependent and T cell-independent antigens, lambs were inoculated twice with rabbit RBC and Escherichia coli lipopolysacharide (LPS) and then were infected with a cytopathic strain of BVDV at postinoculation day 3. Leukopenia characterized by lymphopenia developed after BVDV infection. Increased [3H]thymidine incorporation was observed in resting or lectin-stimulated blood mononuclear cells in the first weeks after inoculation in BVDV-infected lambs, but was followed by decreased [3H]thymidine incorporation after the second inoculation for up to 8 weeks after initial inoculation. In contrast, transient decrease of blastogenic responses, associated with toxic effect of LPS, was detected in inoculated noninfected lambs, but was followed by stimulation of cellular immune responses. Inoculated noninfected lambs had good in vitro cellular immune response to rabbit RBC and LPS antigens, whereas lymphocytes from BVDV-infected lambs could not mount lasting cellular immune responses to antigens or BVDV. Results suggest that BVDV infection in lambs modulates the ability of lymphocytes to respond to lectins or antigenic stimuli according to the time after infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle

There are no pathognomonic clinical signs of infection with bovine viral diarrhoea virus (BVDV) in cattle. Diagnostic investigations therefore rely on laboratory-based detection of the virus, or of virus-induced antigens or antibodies in submitted samples. In unvaccinated dairy herds, serological testing of bulk milk is a convenient method for BVDV prevalence screening. Alternatively, serological testing of young stock may indicate if BVDV is present in a herd. In BVDV positive herds, animals persistently infected (PI) with BVDV can be identified by combined use of serological and virological tests for examination of blood samples. ELISAs have been used for rapid detection of both BVDV antibodies and antigens in blood, but should preferably be backed up by other methods such as virus neutralization, virus isolation in cell cultures or amplification of viral nucleic acid. Detailed knowledge of the performance of the diagnostic tests in use, as well as of the epidemiology of bovine virus diarrhoea is essential for identification of viremic animals in affected herds.     

Infection of ovine fetal brain cell cultures with cytopathogenic and non-cytopathogenic bovine viral diarrhoea virus.

The in vitro cell tropism of non-cytopathogenic (ncp) and cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) was studied in primary dissociated brain cell cultures derived from ovine fetuses of different gestational ages. The cell types infected were identified by double immunofluorescence using antibodies against BVDV and cell type-specific markers. In cultures infected with ncp BVDV viral antigen was present in neurofilament (NF 200 kDa)-positive neurons, glial fibrillary acidic protein (GFAP)-positive astrocytes and fibronectin-expressing cells. Estimation of the percentages of individual cell types infected with ncp BVDV indicated a tropism for NF 200-positive neurons. In cultures infected with cp BVD virus cytopathic changes were observed beginning at 40 hours post infection. Viral antigen was present in vacuolated NF 200-, GFAP- and fibronectin-positive cells. In comparison with non-infected control cultures a considerable reduction of the number of the different cell types was seen.     

Prevalence of bovine viral diarrhea virus (BVDV) in persistently infected cattle and BVDV subtypes in affected cattle in beef herds in south central United States

The prevalence of bovine viral diarrhea virus (BVDV) in persistently infected (PI) cattle in beef breeding herds was determined using 30 herds with 4530 calves. The samples were collected by ear notches and tested for BVDV antigens using immunohistochemistry (IHC) and antigen capture enzyme-linked immunosorbent assay (ACE). Animals with initial positives on both IHC and ACE were sampled again using both tests and serums were collected for viral propagation and sequencing of a viral genomic region, 5′-untranslated region (5′-UTR) for viral subtyping. Samples were also collected from the dams of PI calves. There were 25 PI calves from 4530 samples (0.55%) and these PI calves were from 5 of the 30 herds (16.7%). Two herds had multiple PI calves and 3 herds had only 1 PI calf. Only 1 of the 25 dams with a PI calf was also PI (4.0%). The subtype of all the PI isolates was BVDV1b. Histories of the ranches indicated 23 out of 30 had herd additions of untested breeding females. Twenty-four of the 30 herds had adult cowherd vaccinations against BVDV, primarily using killed BVDV vaccines at pregnancy examination.     

Infectivity of pestivirus following persistence of acute infection

Bovine viral diarrhoea virus (BVDV) is an endemic pathogen worldwide and eradication strategies focus on the identification and removal of persistently infected (PI) animals arising after in utero infection. Despite this, acute infections with BVDV can persist for months or years after the removal of the PI source despite repeated screening for PIs and tight biosecurity measures. Recent evidence for a prolonged duration of viraemia in the testicles of bulls following acute BVDV infection suggests the possibility of a form of chronic persistence that may more closely resemble the persistence strategies of hepatitis C virus (HCV). To investigate the potential for virus transmission from infected and recovered cattle to virus naïve hosts we established an acute infection of 5 BVDV-naïve calves and monitored animals over 129 days. Infectious BVDV was detected in white blood cells between days 3 and 7 post-challenge. The animals seroconverted by day 21 post-infection and subsequently were apparently immune and free from infectious virus and viral antigen.Animals were further monitored and purified white blood cells were stimulated in vitro with phytohaemagglutinin A (PHA) during which time BVDV RNA was detected intermittently.Ninety-eight days following challenge, blood was transferred from these apparently virus-free and actively immune animals to a further group of 5 BVDV-naïve calves and transmission of infection was achieved. This indicates that BVDV-infected, recovered and immune animals have the potential to remain infectious for BVDV-naïve cohorts for longer than previously demonstrated.     

Effects of incubation temperatures and bovine viral diarrhea virus immune serum on bovine turbinate cells persistently infected with a noncytopathogenic isolate of bovine viral diarrhea virus

Noncytopathogenic bovine viral diarrhea virus (BVDV) on initial inoculation in receptive cells induced a persistence in vitro at incubation temperatures of 33, 37, and 39 C. The mechanisms of this persistence were not the result of the induction of defective interfering particles or the selection of temperature-sensitive mutants, but were the result of a naturally occurring noncytopathogenic isolate of BVDV. Persistently infected cells were not freed of the infection by continuous passage in media containing homologous antibody. Persistently infected cells appeared to undergo an earlier senescence than did noninoculated cells of the same passage level and age. This phenomenon was reversed by renewal of the culture media.     

Immunohistochemical and histopathological study of experimental rabies infection in mice.

An immunohistochemical and histopathological study using the ABC technique was carried out to examine time-sequential virus spread in the central nervous system (CNS) of mice after inoculation with the CVS strain of fixed rabies virus by different routes; intracerebral (ic), intraocular (io), intranasal (in), intramuscular (im) and subcutaneous (sc). Only the ic and io inoculations caused fatal infections, so that detailed analysis was conducted on mice inoculated by these two routes. In ic-inoculated mice, viral antigens were detected mainly in neurons in the cerebral cortex and in the pyramidal cells and granular cells of the hippocampus. After io inoculation, viral antigen was first detected in the trigeminal nerve ganglia, following which it spreads to the cerebral cortex and cerebellum. In the hippocampus only a few cells were viral antigen-positive at the early stage after io inoculation. There were no inflammatory lesions or Negri bodies in the CNS of mice infected by either route. This suggests that clinical signs such as ataxia or depression leading to death may be due to the direct effect of the virus on the functions of neural cells, but not to inflammatory reactions. The ABC method will be useful for the early diagnosis of suspected patients or animals to have the disease when conventional histopathological and immunofluorescent antibody techniques can not detect lesions or viral antigens.     

Seroprevalence and factors associated with bovine viral diarrhea virus (BVDV) infection in dairy cattle in three milksheds in Ethiopia

This work was conducted to estimate the seroprevalence, to identify potential factors that influence seroprevalence of bovine viral diarrhea virus (BVDV), and to investigate the association between BVDV serostatus and occurrence of reproductive disorders in dairy cattle in three milksheds in Ethiopia. A total of 1379 serum samples were obtained from cattle randomly selected from 149 herds from three milksheds representing central, southern, and western Ethiopia. Sera samples were examined for bovine viral diarrhea virus (BVDV) antibodies using commercial competitive enzyme-linked immunosorbent assay (ELISA) kit. Logistic regression analysis was employed to investigate associations between risk factors and the risk of BVDV seroprevalence, and BVDV serostatus and reproductive disorders. Seroreaction to BVDV antigens was detected in 32.6% of the 1379 cattle and 69.8% of the 149 herds sampled. Factors associated with BVDV seroplevalence were age, breed, and herd size (P?<?0.05). Adult cattle ≥?18 months old had 2.1 (95% CI 1.5, 3.1) times the odds of BVDV seroreaction than younger cattle. Holstein-Friesian (HF) local crosses (OR?=?2.1, 95% CI 1.3, 3.4) and HFs (OR?=?1.3, 95% CI 0.9, 1.9) were more likely to be seropositive than Jersey and the odds of seropositivity in cattle in large herds with 11 or more animals were higher (OR?=?1.8, 95% CI 1.3, 2.5) than the odds of BVDV seropositivity in smaller herds. Seroprevalence was not associated with geographical region (P?>?0.05). Risk of reproductive disorders was not affected by BVDV serostatus, except for repeat breeding (P?>?0.05). The present study demonstrated that BVDV has wide distribution in the country being detected in all the 15 conurbations and 69.8% of herds involved in the study.     

Human and ovine lentiviral infections compared

Maedi-visna virus (MVV) of sheep was the first lentivirus to be isolated. The genomic organization of MVV is very similar to that of human immunodeficiency virus (HIV) with several genes regulating the expression of the viral genome. Viral replication is severely restricted in the host and some cells apparently contain the genetic information in a DNA provirus form with little or no expression of viral antigens. This seems to be a major factor in causing the “slowness” of lentiviral infections and the persistence of the virus in the host since the immune system may not recognize the provirus-containing cells. The target cells for HIV and MVV are similar although T4 lymphocytes are not specifically destroyed in maedi-visna. There are also certain similarities in the pathological changes in both diseases, both in the central nervous system, the lungs and the lymphatic system. Although the severe final immunodeficiency state characteristic of AIDS has not been observed in maedi-visna, the basic biological features of the MVV and its interaction with host cells are so similar to HIV infection, that we consider ovine maedi-visna useful animal model for the human lentivirus infections.     

Immunohistochemistry used as a screening method for persistent bovine viral diarrhea virus infection.

Cattle persistently infected (PI) with bovine viral diarrhea virus(BVDV) are a major source of infection to herds. To successfully control BVDV, it is necessary to identify and cull those cattle PI with BVDV. Immunohistochemistry (IHC) is a useful tool for sensitive and specific detection of BVDV antigens in infected cattle.Skin of cattle PI with BVDV is one of the tissues where BVDV can be consistently identified by IHC and is readily accessible for sampling. Use of IHC on skin biopsies (in the form of ear notches)as a method to identify cattle PI with BVDV has resulted in a reliable, affordable technique for mass testing of cattle at an early age without maternal antibody interference. The ability to test large numbers of cattle to identify those Pl with BVDV will enable implementation of programs for control and eventual eradication of BVDV.     

Evaluation of the effects of long-term storage of bovine ear notch samples on the ability of 2 diagnostic assays to identify calves persistently infected with bovine viral diarrhoea virus

Research aimed at optimising diagnostic laboratory procedures is central to the development of effective bovine viral diarrhoea virus (BVDV) control programmes. BVDV is a single-stranded RNA virus that crosses the placenta to infect foetuses, resulting in reproductive losses due to foetal death or persistently infected calves that die early in life. Persistently infected animals are widely accepted to be the primary reservoir of BVDV and the largest source of infection. This poses important challenges to overall animal/herd health and can cause major losses to the cattle industry. Long-term storage of bovine ear notch samples from calves persistently infected with BVDV may adversely affect the ability of diagnostic assays to detect the virus efficiently. In order to test this hypothesis, ear notch samples from 7 animals were divided into 2 groups. One set was subjected to prompt formalin fixation and the other set stored either as fresh samples without preservatives at -2 degrees C, or soaked overnight in phosphate buffered saline followed by freezing of the supernatant fluid at -2 degrees C. Frozen ear notches and ear notch supernatant yielded positive results with an antigen-capture, enzyme linked immunosorbent assay (AC-ELISA) for the duration of the study (6 months) and optical density (OD) values remained significantly within range. There was no significant difference between storing fresh ear notch samples or PBS at -2 degrees C. However, positive immunohistochemistry (IHC) staining on formalin fixed ear notches started to fade between Day 17 and Day 29 when stored at room temperature. It was concluded that fresh ear notches could safely be stored at -2 degrees C for a period of 6 months prior to testing for BVD viral antigens.     

Bovine Viral Diarrhea Virus in Swine: Neutralizing Antibody in Naturally and Experimentally Infected Swine

Neutralizing antibodies against bovine viral diarrhea virus (BVDV) were detected in sera of Iowa farm sows, their piglets derived by hysterectomy and in specific-pathogen-free (SPF) pigs in a closed herd. No antibodies against BVDV were detected in sera of 134 fetal pigs from 67 sows in an Iowa slaughter-house. Neutralizing antibodies against BVDV or hog cholera virus, produced experimentally in SPF pigs, were demonstrated to be due to different antigens rather than to a common antigen, although slight cross-neutralization reactions sometimes occur.     

Cytopathic bovine viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction

Bovine viral diarrhea virus (BVDV), a Flaviviridae pestivirus, is arguably one of the most widespread cattle pathogens worldwide. Each of its two genotypes has two biotypes, non-cytopathic (ncp) and cytopathic (cp). Only the ncp biotype of BVDV may establish persistent infection in the fetus when infecting a dam early in gestation, a time point which predates maturity of the adaptive immune system. Such fetuses may develop and be born healthy but remain infected for life. Due to this early initiation of fetal infection and to the expression of interferon antagonistic proteins, persistently infected (PI) animals remain immunotolerant to the infecting viral strain. Although only accounting for some 1% of all animals in regions where BVDV is endemic, PI animals ensure the viral persistence in the host population. These animals may, however, develop the fatal mucosal disease, which is characterized by widespread lesions in the gastrointestinal tract. Cp BVD virus, in addition to the persisting ncp biotype, can be isolated from such animals. The cp viruses are characterized by unrestrained genome replication, and their emergence from the persisting ncp ones is due to mutations that are unique in each virus analyzed. They include recombinations with host cell mRNA, gene translocations and duplications, and point mutations. Cytopathic BVD viruses fail to establish chains of infection and are unable to cause persistent infection. Hence, these viruses illustrate a case of “viral emergence to extinction” – irrelevant for BVDV evolution, but fatal for the PI host.     

Distribution and cellular heterogeneity of bovine viral diarrhea viral antigen expression in the brain of persistently infected calves: a new perspective

Persistent infection following in utero exposure to bovine viral diarrhea virus (BVDV) early in gestation is a serious cause of morbidity and mortality in cattle industries worldwide. The brain is a primary target of persistent infection. In the current study, the types of cells infected and topography of viral antigen expression were examined in brain sections from 9 BVDV persistently infected crossbred calves, all less than 1 year of age, by immunohistochemical staining using the 15C5 primary monoclonal antibody. BVDV antigen was detected in the brains of all persistently infected calves. A variety of cell types was infected, including neurons, astrocytes, oligodendroglia, blood vessel-associated cells (pericytes, perivascular macrophages, smooth muscle cells), and cells in the leptomeninges (blood vessel-associated cells). Conclusive demonstration of viral antigen in vascular endothelial cells was elusive. The intensity and distribution of viral antigen staining in neurons were highly variable. Viral antigen staining was most consistent and intense in thalamic nuclei, most notably in dorsal and medial nuclear groups, followed by the hippocampus, entorhinal cortex, basal nuclei, and piriform cortex. Staining in other brain areas was often less intense and inconsistent. The variability in the intensity and topography of viral antigen in the brain may explain the heterogeneity in the clinical manifestations of BVDV-induced disease. Additionally, infection of the brain in persistently infected calves may underlie or at least contribute to endocrine disturbances and immunologic deficits that are protean manifestations of BVDV-induced disease.