Monoclonal antibodies to bovine viral diarrhea virus: cross-reactivities to field isolates and hog cholera virus strains.

Monoclonal antibodies to bovine viral diarrhea virus (BVDV) were examined for binding with a large number of North American BVDV isolates and eight strains of the serologically related pestivirus, hog cholera virus (HCV). No single BVDV monoclonal antibody reacted with all BVDV isolates. The most cross-reactive monoclonal antibody was an anti-p80/p125 antibody which showed a positive reaction with 173 of 180 (96%) North American isolates. From a fewer number of isolates tested, one anti-gp53 monoclonal antibody also showed a high cross-reactivity (94%). All BVDV isolates showed a positive reaction with at least one of the seven monoclonal antibodies in the panel. Thus, the results indicated that a pool of these monoclonal antibodies may be used in place of polyclonal antisera for the detection of BVDV contamination of cell lines or for virus isolation. For HCV, all three anti-p80/p125 monoclonal antibodies reacted positively with all eight virus strains. In contrast, none of the anti-gp53 monoclonal antibodies were reactive to HCV strains. Thus, the anti-gp53 monoclonal antibodies may be useful for distinguishing between usually innocuous BVDV infections and the highly significant HCV infections in swine for foreign animal disease surveillance.     

Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies

Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared. They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus. A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin. All the MAbs were also tested against representative strains of BVDV and border disease virus. Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses. Among the other HCV MAbs geographical variation in reaction patterns was observed. There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.     

The development of an international reference panel of monoclonal antibodies for the differentiation of hog cholera virus from other pestiviruses.

A panel of 30 monoclonal antibodies was defined and characterized with respect to the binding capacity in immunoperoxidase assay to different strains of pestivirus. Using the panel it was possible to identify specifically all strains and isolates of hog cholera virus, hog cholera vaccines derived from 'C' strains, and most strains of bovine viral diarrhoea/border disease (BVD/BD) viruses (including those isolated from pigs). A small proportion of BVD/BD isolates from pigs and ruminants reacted only with the monoclonals specific for pestivirus group antigen. It is recommended that monoclonal typing methods be introduced into official procedures for the diagnosis of hog cholera/classical swine fever.     

Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm.]

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.     

Detection of border disease virus in a sheep flock in Saxony

A pestivirus has been isolated from brain samples of two lambs suffering from clinical signs of border disease. The two identical isolates were allocated to the "true" border disease virus group concerning their reaction pattern with monoclonal antibodies and the 5'UTR sequence data. Nevertheless, alterations of phenotype and genotype in comparison with references of both BDV-subgroups have been shown. The existence of an infection with border disease virus in the flock has been confirmed by serological studies.     

Bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine

On eight farms a congenital pestivirus infection in piglets was detected which could be traced to vaccination of the dams against swine fever (SF). Viruses isolated from the piglets were not recognised by monoclonal antibodies (MCAs) against swine fever virus (SFV) and were shown to be bovine viral diarrhoea virus (BVDV) or Border disease virus. The expected 'Chinese' strain of SFV could not be demonstrated in the batch of vaccine that had been used on these farms. Instead, a contaminating pestivirus was recovered which was not recognised by the MCAs against SFV. The contaminant had an unexpectedly high virulence for lambs and induced antibodies against BVDV in lambs and pigs. It could, therefore, be characterised as BVDV or Border disease virus.     

Genetic diversity and BVD virus.

The various measures of genetic variation of BVD virus was reviewed with emphasis on the implications for future control of virus-induced disease and diagnosis. While experimental data does not support unique serotypes for BVDV, there is substantial antigenic variation among the isolates examined. This variation may permit fetal infections even in animals assumed to be well vaccinated. The genetic differences between cytopathic and noncytopathic strains of BVDV are expressed in infected cells by the production of a p80 protein by cytopathic strains. In addition, cellular gene inserts have been detected in cytopathic strains. Monoclonal antibodies have demonstrated a high degree of diversity with the pestivirus population. Grouping of BVDV isolates by monoclonal antibody analysis is suggestive at best. The use of nucleic acid probes as diagnostic reagents has been compromised by the nucleic acid sequence variation found in the BVDV isolates tested.     

Monoclonal antibodies to the p80/125 gp53 proteins of bovine viral diarrhea virus: their potential use as diagnostic reagents.

Monoclonal antibodies reactive to the bovine viral diarrhea virus (BVDV) protein gp53 were produced and characterized. These antibodies and our panel of anti-p80/125 monoclonal antibodies were tested for their cross-reactivity with 11 different North American and European (Danish) BVDV strains and isolates including viruses of both cytopathic and noncytopathic biotypes. The four anti-gp53 monoclonal antibodies were neutralizing for the homologous Danish cytopathic isolate and cross-reacted with all BVDV strains examined except for the Draper strain. Further, anti-gp53 monoclonal antibodies neutralized the majority of BVDV strains examined. The anti-p80/125 monoclonal antibodies cross-reacted with all eleven strains and isolates tested. This indicated that various strains of BVDV have common epitopes. The broad cross-reactivities demonstrated by these monoclonal antibodies suggest that a pool of these antibodies may be used for detection of BVDV cellular contamination or for virus isolation, in place of polyclonal antiserum.     

Pathomorphological and immunohistological findings in progeny of goats experimentally infected with pestiviruses.

A total of 25 pregnant goats without neutralizing antibodies against BVD virus were inoculated with two different pestivirus isolates at eight different stages of gestation. In both infection groups, various malformations were observed in fetuses and neonates. In three twins with neutralizing antibodies against BVD virus leukoencephalomalacia occurred, characterized by gelatinous transformation in the cerebral hemispheres. These lesions were comparable to alterations described in alternative pathology of Border disease in sheep. Although the immunohistological findings are characteristic for immunological tolerance and viral persistence, viable offspring persistently infected with pestivirus was not observed.     

Comparative analysis of monoclonal antibodies against pestiviruses: report of an international workshop

Thirty-three pestivirus strains were grown in cell culture and characterized by immunostaining with 19 monoclonal antibodies (MAbs) raised against hog cholera virus (HCV), with 42 MAbs against bovine viral diarrhoea virus (BVDV) and with 13 MAbs against border disease virus (BDV). Seven MAbs reacted with all pestivirus strains tested, eight MAbs detected only the seven HCV strains, three detected only the 16 BVDV strains. No MAb was found that was specific for BDV. BVDV and BDV strains were broadly cross-reactive with the MAbs, indicating a close relationship between these two species, whereas HCV strains were characterized as distinct from BVDV and BDV.     

Antigenic relationship of turkey coronavirus isolates from different geographic locations in the United States

The purpose of the present study was to examine the antigenicity of turkey coronavirus (TCV) isolates from various geographic areas with antibodies to different viruses. Seventeen isolates of TCV were recovered from intestinal samples submitted to Animal Disease Diagnostic Laboratory, Purdue University, from turkey farms located in different geographic areas. The prototype TCV Minnesota isolate (TCV-ATCC) was obtained from the American Type Culture Collection. Intestinal sections were prepared from turkey embryos infected with different TCV isolates and reacted with polyclonal or monoclonal antibodies to TCV, infectious bronchitis virus (IBV), bovine coronavirus (BCV), transmissible gastroenteritis virus (TGEV), reovirus, rotavirus, adenovirus, or enterovirus in immunofluorescent antibody staining. All 18 TCV isolates have the same antigenic reactivity pattern with the same panel of antibodies. Positive reactivity was seen with polyclonal antibodies to the TCV Indiana isolate, the TCV Virginia isolate, TCV-ATCC, and the IBV Massachusetts strain as well as monoclonal antibodies to the TCV North Carolina isolate or the membrane protein of IBV. Antibodies to BCV or TGEV were not reactive with any of the TCV isolates. Reactivity of antibodies to unrelated virus, rotavirus, reovirus, adenovirus, or enterovirus with different TCV isolates was all negative, except positive response was seen between enterovirus antibody and a TCV western North Carolina isolate, suggesting coinfection of turkeys with TCV and enterovirus in that particular case. The results indicated that the TCV isolates from these geographic locations in the U.S. shared close antigenicity and were antigenically related to IBV.     

Serological evidence for exposure to bovine viral diarrhoea virus in sheep co-grazed with beef cattle in New Zealand

ABSTRACT

Aims: To determine whether sheep that co-grazed with cattle that were suspected to be positive for bovine viral diarrhoea (BVD) virus had serological evidence of exposure to the virus.

Methods: Eighteen commercial farms that routinely co-grazed cattle and sheep in the same paddocks were recruited through purposive sampling. The recruiting veterinarians identified nine farms with cattle herds that were known or highly suspected to be positive for BVD and nine farms that were considered to be free of BVD. Blood samples were taken from 15 ewes aged 1 year on each farm and samples were submitted to a commercial diagnostic laboratory to test for antibodies against pestiviruses using an ELISA. All samples that were positive were then tested using a virus neutralisation test (VNT)for antibodies against BVD virus.

Results: Of the 270 blood samples, 17 were positive for pestivirus antibodies by ELISA and these originated from two farms that were known or suspected to have BVD virus-positive cattle. None of the samples from the nine flocks co-grazed with cattle herds that were known or suspected to be BVD virus-negative were positive for pestivirus antibodies. Within the two positive farms, 2/15 samples from the first farm and 15/15 samples from the second farm were antibody-positive. When the 17 positive blood samples were submitted for VNT, all 15 samples from the second farm tested positive for BVD virus antibodies with the highest titre being 1:512.

Conclusions and clinical relevance: In this small sample of New Zealand sheep and beef farms with suspected BVD infection in cattle, there was evidence of pestivirus exposure in co-grazed sheep. Although we were unable to confirm the origin of the exposure in these sheep, these findings highlight that farmers who are trying to eradicate BVD from their cattle should be mindful that the infection may also be circulating in sheep, and both populations should be considered a possible risk to each other for generating transient and persistent infections. Further work is needed to estimate the true prevalence of New Zealand sheep flocks that are affected by BVD and the associated economic impacts.     

Differentiation of parapoxviruses by application of orf virus-specific monoclonal antibodies against cell surface proteins.

Monoclonal antibodies were produced against orf virus-specified cell surface proteins in an attempt to develop reagents capable of differentiating between members of the Parapoxviridae. Two immunization protocols were used to induce an anti-orf response in BALB/c mice, one of which resulted in virus replication in the recipient. The monoclonal antibodies produced were tested for crossreactivity with bovine papular stomatitis virus (BPS) and milker's node virus (MNV) by indirect immunofluorescence assay (IFA) and immunoblotting. The results indicate that significant antigenic overlap exists between isolates of orf, MNV and BPS, even at the level of specificity provided by monoclonal antibodies. One monoclonal antibody reacted strongly in IFA with orf virus isolates, very weakly with MNV, and not at all with BPS. On immunoblots this same antibody recognized a 40-43 kDa protein in orf virus-infected cells, and also a 45-48 kDa protein in cells infected with MNV or BPS virus. The data suggest that it may be possible to define parapoxvirus strains on the basis of small variations in specific virus-directed cell surface proteins.     

Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses

A collection of 90 field isolates of hog cholera virus (HCV) was used to test the specificity of four hybridoma cell lines secreting monoclonal antibodies against pestiviruses. Reaction of virus isolates and monoclonal antibodies was controlled by an indirect immunofluorescence assay (IFA). Two monoclonal antibodies which had been generated against HC virus strain "Alfort 187" were reactive only with HCV field isolates and an HCV reference strain but not with bovine viral diarrhoea virus (BVDV) reference strains. Two other monoclonal antibodies (generated against BVDV, strain NADL) reacted only with BVDV reference strains but not with HCV field isolates, although with 3 of these strains focal reactions involving only a few cells were detected. The ability to discriminate between both viruses is a diagnostic need which may be fulfilled by these monoclonal antibodies.     

Similarity of avian paramyxovirus serotype 1 isolates of low virulence for chickens obtained from contaminated poultry vaccines and from poultry flocks

At present Denmark has the status of a 'non-vaccinating' country for Newcastle disease and its poultry population should therefore be free of antibodies to avian paramyxovirus 1 (APMV-1). Three live avian vaccines against infectious bronchitis, avian encephalomyelitis, and chick anaemia which had been found to be contaminated with APMV-1 viruses of low virulence for chickens were examined. The vaccines were produced by the same company and the affected batches had been used in Denmark in 1996/97. Furthermore, APMV-1 isolates of low virulence were obtained from three commercial broiler breeder flocks, one of which had been vaccinated with two of the contaminated vaccines. The flocks belonged to the same hatchery organisation. A comparison of viral F0 gene sequences and typing of virus isolates with a panel of monoclonal antibodies showed that the vaccine and field isolates were identical.     

In vitro and in vivo association of African swine fever virus with swine erythrocytes

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.     

An outbreak of mucosal disease in a dairy herd

An outbreak of mucosal disease (MD) was studied in a dairy herd, comprising 12 cows, 9 heifers and 18 calves. During a period of 1 month, six 5 to 8 month-old calves showed typical signs of MD. They all died or were killed in extremis after 2-8 days with progressively worsening clinical signs. Post mortem lesions were examined in one calf. Non-cytopathogenic MD virus was isolated from serum or tissues from 3 clinically affected calves and from 1 healthy heifer. All cows and heifers except for the viremic one possessed neutralizing antibodies against bovine pestivirus. According to the current MD-pathogenesis concept, the affected calves were probably infected transplacentally during the first half of foetal life with pestivirus from the persistently infected heifer in the herd.     

Isolation and characterization of bovine Theileria parasites in Zimbabwe

Theilerial parasites of cattle were isolated by a variety of methods from the Harare area of Zimbabwe. Parasite stocks were established in lymphoid cell cultures and as cryopreserved sporozoite stabilates in the laboratory. Fourteen stocks in culture were characterized by testing them with monoclonal antibodies (MAb) raised against T. parva parva and T. parva lawrencei antigen. Two of these stocks had profiles similar to T. taurotragi isolates from East Africa, the other stocks had profiles similar to T. parva parva, however, many of them failed to bind MAb No. 7, and this may be a distinctive feature for T. parva bovis. Three T. p. bovis stocks were titrated by injecting different doses of the respective stabilates into pairs of cattle. Reactions ranged from severe to inapparent according to the stocks and dose used, but no fatal reactions were recorded, even at the highest dose rate. On recovery, all cattle were given homologous and then heterologous challenge. The results of the latter challenge showed that the Boleni stock gave good cross-protection against challenge with two other Zimbabwean stocks. This stock may therefore be a candidate for immunizing cattle, under field conditions, to protect them against T. p. bovis in Zimbabwe. Non-pathogenic strains of T. p. bovis may be difficult to distinguish from T. taurotragi unless cross-challenge experiments can be conducted and/or MAb profiles have been made. An improved serological test is needed to differentiate antibodies to these parasites in the sera of recovered cattle.