Leon N. D. Potgieter

Immunology of Bovine Viral Diarrhea Virus

Often persistent and primary postnatal infections with BVDV result in immunosuppression in cattle, thereby enhancing the vulnerability of the latter to secondary infections. The evidence for and nature of impaired immunity in these animals is reviewed. Our knowledge of the extent and nature of protective immunity induced by natural BVDV infections and vaccines is still in its infancy. Significant new information on the bovine immune response to BVDV antigens, however, has been identified in recent years. These data are reviewed and related to prospects for improved immunoprophylaxis against diseases caused by the virus.

Bovine Respiratory Tract Disease Caused by Bovine Viral Diarrhea Virus

Although several viruses and bacteria are capable of inducing bovine respiratory tract disease, a pivotal organism in the cause of this complex disease may be bovine viral diarrhea virus (BVDV). Circumstantial evidence has long supported this hypothesis. It is frequently present in diseased respiratory tract tissues often together with other viruses or bacteria. Field observations suggest marked synergism occurs. Researchers have confirmed that, in most instances, the virus itself elicits only a mild respiratory tract disease in susceptible calves, but some strains may be much more pneumo-pathogenic than others. Experimental evidence now supports the hypothesis, that BVDV markedly enhances respiratory tract disease caused by IBRV, BRSV, or Pasteurella haemolytica; that it impairs pulmonary immunity; and that it, by itself, may produce mild respiratory tract disease.

A serologic survey of selected viral and bacterial diseases of European wild hogs, Great Smoky Mountains National Park, USA.

Blood samples were collected from 108 wild hogs (Sus scrofa) from the Great Smoky Mountains National Park (GSMNP), USA, February to July 1990. We found no antibodies for swine brucellosis, pseudorabies, bovine virus diarrhea virus or porcine rotavirus infection. Antibody titers to porcine parvovirus were found in 15 (14%) samples and antibody to one or more leptospiral serovars was found in 48 (44%) samples. Thirty-nine (89%) of the 44 positive samples reacted to all five leptospiral serovars tested.

Single and Double Polymerase Chain Reaction for Detection of Bovine Viral Diarrhea Virus in Tissue Culture and Sera

Bovine viral diarrhea virus (BVDV) is an ubiquitous pathogen of cattle and has been reported in other ruminants. It is also frequently present in laboratory and biological materials as an adventitious agent. This virus is difficult to detect in some specimens, especially in the presence of specific antibody and when the virus is present in low concentrations. In this paper, we describe a single polymerase chain reaction (PCR) to amplify virus sequences from infected cell culture and a nested double PCR to detect small concentrations of several virus strains in sera. Total cellular RNA was extracted from cell cultures infected with the cytopathic strain 72 and noncytopathic strain 2724 of BVDV. Ten different genomic sequences along the length of the viral RNA ranging in size from 397 to 1,016 base pairs (bp) were successfully amplified by PCR. A 404-bp probe made from amplified product from the 3′ end hybridized specifically with the RNA of several BVDV strains blotted on nylon filters. Viral RNA was extracted from serum and amplified using 2 sets of degenerate nested primers designed from the 3′ end of the viral genome in a double PCR protocol. Double amplification of the viral sequences greatly enhanced the sensitivity of the detection of many strains present in serum. Advantages of using double PCR over single PCR and virus isolation is discussed.

Antigenic diversity of respiratory syncytial viruses and its implication for immunoprophylaxis in ruminants

Bovine respiratory syncytial virus (BRSV) is a very important pathogen of cattle and perhaps other ruminants. It is a major contributor to the incidence of respiratory tract disease in nursing beef and feedlot and dairy calves. The genome of respiratory syncytial viruses encodes 10 proteins translated from 10 unique mRNAs. The major glycoprotein (G), fusion protein (F), 1A protein and the 22K protein are components of the viral envelope. The nucleocapsid contains the nucleocapsid protein (N), the phosphoprotein (P), and the large protein (L). The matrix protein (M) forms a structural layer between the envelope and the nucleocapsid. Antibodies to all the structural proteins develop in convalescent calves. However, evidence suggests that immunity develops primarily as a result of the antigenic stimulus by the major glycoprotein G and the fusion glycoprotein F. It is known also that activated cytotoxic T cells interact with N and F protein antigens and helper T cells interact with N, F, and 1A protein antigens. With the exception of the major glycoprotein, the respective proteins of various respiratory syncytial viruses share major antigenic domains. Based on antigenic differences of the major glycoprotein, at least 3 subgroups of RSV are recognized; human A, human B, and bovine RSV. Indirect evidence suggests that a second subgroup of BRSV exists. However, we have identified only one BRSV subgroup based on our work with RNase mismatch cleavage analysis of the G protein gene from a limited number of strains. Furthermore, our data indicated that a caprine RSV isolate is closely related to the bovine strains, but an ovine isolate is not. The latter may constitute yet another subgroup of RSV. These data affect decisions on optimization of immunoprophylaxis since evidence suggests that protection against a homologous RSV subgroup virus is superior to that against a heterologous strain in immune subjects.

Genetic Analysis of Feline Caliciviruses Associated with a Hemorrhagic-Like Disease

Feline calicivirus (FCV) is 1 of the most common causes of upper respiratory tract disease in cats. Other disease syndromes associated with FCV infection have been reported. Recently, calicivirus infection associated with a hemorrhagic-like disease leading to significant mortality in cats has been reported. The clinical signs are similar to those observed with the calicivirus of rabbit hemorrhagic disease. This study characterized 2 FCV isolates associated with hemorrhagic-like disease. Nucleotide sequencing of the complete genome has been done for these 2 isolates as well as for 4 additional isolates representing other disease syndromes. Previously reported sequence data for the entire genome of classical FCV (6 isolates) and a portion of the capsid gene for hemorrhagic-like FCV (3 isolates), isolated in different regions of United States were used in the genetic analysis. Sequence data were used to determine relationships among the isolates and any correlation with phenotype. Nucleotide sequence comparisons of the entire genome and individual open reading frames revealed high homology among all isolates. Data suggest that the virulence may have genetic determinants on the basis of phylogenetic clustering of the isolates associated with hemorrhagic-like disease.

Correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal disease of domestic and nondomestic felids caused by a feline coronavirus (FCV). It is a significant problem in catteries, multiple cat households, and shelters. 14,18 FIP can manifest as an effusive peritonitis and/or pleuritis, with a relatively short disease course ending in death. A protracted course with granulomatous lesions affecting multiple organs may occur, which also invariably progresses to death. 8,11,12 The FCVs are closely related and include two biotypes: those that are virulent and cause FIP and those that are avirulent. 7 The avirulent group, known as feline enteric coronaviruses (FECV), may be associated with mild enteric disease or subclinical infection in cats. 11,12 FCVs are also classified into serotypes 1 and 2 based on antigenicity. 7,12 Both serotypes contain virulent and avirulent biotypes, and FCVs within a serotype are indistinguishable in the laboratory. FCVs are ubiquitous, especially in environments such as catteries, where large numbers of cats are housed together and where the majority of cats are seropositive. 12 As a result, antemortem diagnosis for FIP is difficult because no detection assay sensitive enough and specific for the virus causing FIP is routinely available. 12 This problem is further compounded because consistent genetic differences between the virulent and avirulent biotypes of FCV have not been identified. 16 An assay has been developed using reverse transcription and a nested polymerase chain reaction (PCR) to detect FCV genomic material in a variety of biologic samples from cats. The genomic region targeted encompasses the 7a and 7b open reading frames (ORFs). This region, specifically that encoding the 7b ORF, may correlate with virulence.

An Improved Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Assay for Gender Identification in Birds

Abstract A polymerase chain reaction (PCR) technique, targeting a chromo-helicase DNA-binding protein (CHD) gene, was used to determine gender in birds. DNA from 87 birds, representing 31 species from 9 of the 20 orders of birds, was evaluated. The DNA was extracted from as little as 5 μ l of whole blood, and CHD-specific primers (P2 and P8) were used to amplify the CHD gene. Polymerase chain reaction products were then subjected to restriction fragment length polymorphism analysis. Gender was determined by distinguishing between the female CHD-W gene and the male CHD-Z gene by agarose gel electrophoresis of the digested PCR products. The PCR assay using the HaeIII restriction enzyme helped identify gender in all species evaluated except those from the Order Anseriformes. Isolation of the CHD-Z and CHD-W fragments was performed, and the genes were cloned and sequenced in a number of waterfowl species. On the basis of sequence results for the CHD-Z and CHD-W genes for both the mallard duck (Anas platyrhynchos) and the Canada goose (Branta canadensis), the cleavage site for the enzyme EcoRI was identified, and the lack of a cleavage site for HaeIII was confirmed. EcoRI was then used to sex 5 duck species and the Canada goose.

Serologic reactivity using conserved envelope epitopes in feline lentivirus-infected felids

An enzyme-linked immunosorbent assay (ELISA) based on synthetic peptides identical to lentivirus envelope protein amino acid sequences was used to study serologic reactivity of lentivirus-infected domestic cats and nondomestic felids. One feline immunodeficiency virus (FIV) peptide, P237, was consistently recognized by antibodies from FIV-infected cats, but 2 other FIV peptide antigens were not. The molecular basis for this serologic reactivity was examined. Lentivirus-infected nondomestic Felis species reacted intensely with a puma lentivirus (PLV) peptide corresponding to the conserved FIV peptide. However, lentivirus-infected Panthera species, from which a different lentivirus has been isolated, did not react with the PLV. FIV-infected domestic felids also did not have significant reactivity with the PLV peptide. The peptide ELISA is comparable in sensitivity and specificity to western blot analysis and a commercial enzyme immunoassay. Unlike the other assays, however, the peptide ELISA is inexpensive, requires a small amount of serum, enables the study of specific isotype reactivity, and discriminates between antibodies to FIV and those to PLV. Antibody tests based upon the FIV and the PLV peptides should be useful for detecting the possible introduction of FIV into exotic felids or of lentiviruses from nondomestic felids into the domestic cat population.

Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay

Subgroup-specific peptide-based enzyme-linked immunosorbent assays from the G-protein of the ovine and bovine respiratory syncytial virus (RSV), respectively, were used to determine the prevalence of the ovine and bovine subgroup strains of RSV infections in cattle. A total of 1,102 bovine serum samples were obtained from 6 diagnostic laboratories located in the northwestern and the southeastern USA and were tested for antibody to either the bovine or ovine subgroups of RSV. Antibody to viruses from each subgroup was present in samples from each region and all states tested. The Southeast had a higher prevalence of the bovine subgroup strains (69.5%). Then did the Northwest (40.9%). The prevalence of the ovine strain was similar for the two regions (16.7% in the southeast, 14.9% in the northwest). The overall prevalence was 56.6% for the bovine strain and 15.9% for the ovine strain. These results suggest members of the ovine subgroup of RSV circulate in the cattle population but with less frequency than those viruses of the bovine subgroup.