Bovine herpesvirus-1 infection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus

The kinetics of antibody formation in Holstein heifers after primary and secondary intranasal inoculation of bovine herpesvirus-1 (BHV-1) and after BHV-1-induced abortion was determined. Sera were fractionated by gel filtration and ion-exchange chromatography. The antibody activity within serum immunoglobulin (Ig) isotypes was assessed, using a plaque-reduction neutralization assay. The primary immune response to BHV-1 infection was characterized by the appearance of IgM and IgG antibodies in serum by postinoculation day (PID) 7. Maximal IgG antibody activity occurred at PID 35 in nonpregnant heifers and at PID 14 in pregnant heifers. Thereafter, IgG antibody activity declined slowly in both groups of heifers. Maximal IgM antibody activity occurred at PID 14 in both groups of heifers and declined rapidly thereafter. The IgG antibody activity during primary immune responses was restricted to the IgG1 subclass. Secondary responses were characterized by anamnestic IgG antibody responses. Antibody activity was present within the IgG1 and IgG2 subclasses during secondary immune responses, but the increase in antibody activity during this period was primarily in the IgG2 subclass. Secondary IgM antibody formation was elicited by abortion induced by the intra-amniotic inoculation of BHV-1, but not by reexposure by the intranasal route. Abortion occurred in 1 heifer 28 days after intranasal BHV-1 inoculation. Abortion in this heifer was not associated with a secondary antibody response. The nature of BHV-1 antigenic exposure in the bovine determined the relative distribution of anti-BHV-1 antibody activity in serum IgM, IgG1, and IgG2. The formation of IgM antibody, with the exception of secondary intranasal exposure, indicated recent BHV-1 antigenic exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of bovine herpesvirus-1-specific IgM using a capture enzyme immune assay with isotype-specific monoclonal antibodies

The detection of virus-specific immunoglobulin M (IgM) antibodies in acute-phase serum samples offers the possibility of making an accurate and rapid serologic diagnosis. We have developed a solid-phase capture assay that uses murine monoclonal antibodies specific for bovine IgM to separate the whole IgM fraction of a bovine serum sample. The IgM specific for bovine herpesvirus-1 (BHV-1) is then detected by the addition of viral antigen, which in turn is detected by BHV-1-specific monoclonal antibodies conjugated to horseradish peroxidase. A BHV-1 IgM antibody response was detected during the early postinfection period (7-40 days PI). Bovine herpesvirus-1 IgM antibody was not detected in sera taken from 3 animals following dexamethasone-induced viral reactivation. This method compares favorably with viral isolation, antigen detection in the clinical samples, and paired serology in the diagnosis of BHV-1 infection at a herd level.     

Determination of ability of a thymidine kinase-negative deletion mutant of bovine herpesvirus-1 to cause abortion in cattle.

The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase-negative (TK-) deletion mutant virus. Twelve heifers were inoculated IV at 25 to 29 weeks of pregnancy with either TK- or thymidine kinase-positive (TK+) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of TK+ inoculates were slightly higher and remained above normal a few days longer than in TK- inoculates. Viremia was detected in 5 of 6 TK+ inoculates and in all 6 TK- inoculates. More virus isolations were made from nasal and vaginal swab specimens of TK+ inoculates than from swab specimens of TK- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the TK- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given TK+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the TK gene reduces abortifacient activity of bovine herpesvirus-1.     

Induction of immune responses in cattle with a DNA vaccine encoding glycoprotein C of bovine herpesvirus-1

A DNA vaccine expressing glycoprotein C (gC) of bovine herpesvirus-1 (BHV-1) was evaluated for inducing immunity in bovines. The plasmid encoding gC of BHV-1 was injected six times intramuscularly or intradermally into calves at monthly intervals. After immunization by both routes neutralizing antibody and lymphoproliferative responses developed. The responses in the intradermally immunized calves were better than those in calves immunized intramuscularly. However, the intradermal (i.d.) route was found to be less efficacious when protection against BHV-1 challenge was compared. Following intranasal BHV-1 challenge, all immunized calves demonstrated a rise in IgG antibody titre on day 3, indicating an anamnestic response. The control non-immunized calf developed a neutralizing antibody response on day 7 post-challenge. The immunized calves showed a slight rise in temperature and mild clinical symptoms after challenge. The intramuscularly immunized calves showed earlier clearance of challenge virus compared with intradermally immunized calves. These results indicate that DNA immunization with gC could induce neutralizing antibody and lymphoproliferative responses with BHV-1 responsive memory B cells in bovines. However, the immunity developed was not sufficient to protect calves completely from BHV-1 challenge.     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

Preparation of heavy chain specific antisera to bovine IgA, IgM, IgG1 and IgG2

Goats, guinea pigs and rabbits were immunized with bovine IgM or with intact molecules, heavy chains, Fc portions or light chains of bovine IgG1 and IgG2. Rabbits and guinea pigs were immunized with bovine secretory IgA. Goats and guinea pigs produced heavy chain specific antisera to intact IgM whereas rabbits produced anti-light chain antibody and in one instance anti-alpha 2-macroglobulin antibody in addition to the anti-mu response. Goats and guinea pigs produced antisera to bovine IgG1 and IgG2 and their Fc portions that needed little absorption to render them monospecific for the heavy chain. In addition to antibody to the heavy chains, rabbits produced anti-light chain antibody when immunized with intact IgG1 or IgG2 molecules. These latter sera, and those produced by rabbits immunized with Fc portions of IgG1 or IgG2 required extensive absorption before they were monospecific for their respective heavy chains. Heavy chains were poor immunogens in all three species. Rabbits immunized with IgA produced both anti-alpha and anti-light chain antibodies while guinea pigs produced sera with antibody activity to the alpha chain only.     

Humoral immunity in the ewe. 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.     

A dot immunobinding assay on nitrocellulose for the detection of bovid herpesvirus-4 antibodies

A dot immunobinding assay (DIA) was developed for the detection of antibody against bovid herpesvirus-4 (BHV-4) in bovine sera. A semipurified virus preparation was used as the antigen and an antispecies horseradish peroxidase-labeled IgG and diaminobenzidine were employed as the detection system. The sensitivity and specificity of the DIA were similar to those of indirect fluorescent antibody test, indicating the suitability of DIA as a rapid field test for the detection of anti-BHV-4 antibodies in cattle.     

Evaluation of antibovine IgG subclass specific antisera from guinea pigs and goats

A technique for producing specific antibovine IgG2 antibodies is described. The method relies on the abrogation of the class-specific antibody response of guinea pigs to bovine IgG1 by intravenous injection of goat serum immediately before immunisation in the foot pads with bovine IgG2 in adjuvant. Of the 10 resulting antisera, six were judged monospecific for IgG2 by immunoelectrophoresis but, of these, two antisera gave a very faint line in gel diffusion using IgG1 as the antigen. Radial immunodiffusion studies indicated that the strength of the antisera, using IgG2 as the antigen, was similar to antisera of guinea pigs not injected with goat serum before absorption with bovine IgG1. For guinea pigs injected with goat serum, using bovine IgG1 as an immunogen did not result in the production of subclass specific antisera, rather, the specificities were similar to those of animals not receiving goat serum. This data is compared to absorption studies of goat antibovine IgG1 and IgG2 antisera. The relationships of goat and bovine IgG subclasses are discussed.     

Serological responses of specific pathogen-free foals to equine herpesvirus-1: primary and secondary infection, and reactivation.

Serum antibody (virus neutralisation, complement fixation, IgM and IgG) responses to equine herpesvirus-1 (EHV-1) infection were measured in six foals which were initially free from EHV-1 and EHV-4 infection and maternally-derived antibodies. Following primary infection, high titres of virus neutralisation and complement fixation antibodies were detectable against EHV-1, however, corresponding antibody levels against EHV-4 were low or inapparent, although the two viruses share a number of cross-reactive epitopes. In addition, following the primary infection with EHV-1, IgM levels increased before those of IgG, virus neutralisation and complement fixation antibodies, peaked sooner and thereafter declined. Stimulation of IgM levels was observed on secondary infection with EHV-1 given 61 days later. In contrast, IgG, virus neutralisation and complement fixation antibodies following primary infection were more sustained and no increase in their levels was observed on secondary infection. No consistent changes in IgM or IgG levels were seen after administration of dexamethasone to reactivate latent virus.     

Seroepidemiology of undifferentiated fever in feedlot calves in western Canada.

The relationships between 4 bacterial and 3 viral antibody titers and morbidity (undifferentiated fever (UF)) and mortality were investigated in recently weaned beef calves. Blood samples from 100 animals that required treatment for UF (Cases) and 100 healthy control animals (Controls) were obtained: upon arrival at the feedlot (Arrival), at the time of selection as a Case or Control (Selection), and at approximately 33 d of the feeding period (Convalescent). Seroconversion to Pasteurella haemolytica antileukotoxin was associated with an increased risk of UF (OR = 2.83); however, seroconversion to bovine herpesvirus-1 G-IV glycoprotein was associated with a decreased risk of UF (OR = 0.43). Higher Arrival bovine viral diarrhea virus antibody titer was associated with a decreased risk of UF (OR = 0.83). Increases in Mycoplasma alkalescens antibody titer after Arrival were associated with an increased risk of UF (OR = 1.10). Higher Arrival Haemophilus somnus antibody titer and increases in Haemophilus somnus antibody titer after Arrival were both associated with a decreased risk of UF (OR = 0.76 and OR = 0.78). The odds of overall mortality (OR = 5.09) and hemophilosis mortality (OR = 11.31) in Cases were significantly (P < 0.05) higher than in the Controls. Higher Arrival bovine herpesvirus-1 antibody titer was associated with an increased risk of mortality (OR = 1.30). Protective immunity to Pasteurella haemolytica antileukotoxin, Haemophilus somnus, bovine herpesvirus-1 G-IV glycoprotein, bovine viral diarrhea virus, and Mycoplasma spp. may be necessary to reduce the occurrence of UF. Animals with UF are at an increased risk of overall and hemophilosis mortality.     

Modulation of IgG subclass expression during antibody responses in sheep

Experiments were undertaken to investigate IgG subclass expression during epitope-specific antibody responses in sheep. Animals were immunised with conjugates of bovine serum albumin-DNP (BSA-DNP), or killed Staphylococcus aureus-DNP (Sa-DNP) alone or with muramyl dipeptide (MDP), dextran sulphate (DXS), or staphylococcal exotoxins (toxin). Sheep received two injections of the same preparation intracutaneously at six weeks interval. Total and IgG subclass-specific anti-DNP, and anti-carrier (BSA or S aureus) antibody levels were measured by ELISA in blood taken at weekly intervals before and after immunisation. Anti-DNP antibody levels in animals given Sa-DNP alone were considerably greater than in those immunised with BSA-DNP alone. Toxin suppressed antibody responses to DNP and both carriers; MDP suppressed anti-hapten antibody responses below the levels obtained with antigen alone. Neither toxin nor MDP significantly altered the IgG subclass profile of antibody to DNP bound to either carrier. DXS did not significantly change total levels of anti-DNP antibody measured in sheep given BSA-DNP or S aureus-DNP. However, DXS promoted IgG1 and suppressed IgG2 anti-DNP antibody responses during the secondary response to Sa-DNP but not to BSA-DNP.     

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).     

Comparison of humoral immune responses in dairy heifers vaccinated with 3 different commercial vaccines against bovine viral diarrhea virus and bovine herpesvirus-1

A randomized clinical trial was conducted to compare the humoral immune response to 3 different commercial vaccines in dairy heifers housed in 3 different dairy farms in Quebec. All heifers were seronegative to type 1 bovine viral diarrhea virus (BVDV) (Singer strain), type 2 BVDV (NVSL 125c strain), and bovine herpesvirus-1 (BHV-1) at the beginning of the trial. In addition, control heifers in group 1 remained seronegative to the 2 viruses till the end of the trial. Significant differences in humoral immune responses occurred among the 3 commercial vaccines at 4 weeks and 6 months following vaccination. The vaccine in group 2 elicited higher mean antibody titers and seroconversion rates to both type 1 and type 2 BVDV than that in groups 3 or 4. Vaccines in groups 2 and 3 induced higher mean antibody titers to BHV-1 than did the vaccine in group 4.     

Molecular diagnosis of alcelaphine herpesvirus (malignant catarrhal fever) infections by nested amplification of viral DNA in bovine blood buffy coat specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.     

The possible role of stress in the induction of pneumonic pasteurellosis.

Five groups of range bred calves (four calves per group) were used to investigate the effect of stress on susceptibility to aerosol exposures with bovine herpesvirus-1 or Pasteurella haemolytica. Twelve calves were weaned, transported, processed at a commercial feedlot and transported to isolation facilities three days later. An aerosol challenge of either 10 colony forming units of P. haemolytica or 10 plaque forming units of bovine herpesvirus-1 virus was given to two groups of calves and the third group was not challenged. The fourth group was transported directly to the isolation facilities after weaning and aerosol challenged with P. haemolytica. The fifth group remained at the farm after weaning and was not challenged. All transported animals had elevated plasma cortisol levels which remained above normal for at least three days postchallenge. The blastogenic response of all calves was depressed after leaving the farm and remained depressed throughout the experiment. The suppression correlated well with elevated serum cortisol levels. Calves processed through the feedlot encountered bovine herpesvirus-1 because eight out of 12 animals seroconverted to this antigen. Most calves seroconverted to P. haemolytica whether they were experimentally challenged or not. Where the unchallenged calves encountered P. haemolytica is unknown. Calves challenged with bovine herpesvirus-1 but not with P. haemolytica, had significant clinical signs of pneumonia and two animals died due to bovine herpesvirus-1 infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

Bovine monoclonal antibodies specific for bovine herpesvirus-1 glycoprotein gIII

Spleen cells from a calf immunized with bovine herpesvirus-1 (BHV-1) were fused with the nonsecreting murine cell line SP2/0. Several bovine-murine hybridomas secreting bovine immunoglobulins were stabilized. Of these, 9 hybridomas secreted bovine monoclonal antibodies that specifically bound to BHV-1 in a radioimmunoassay. Two of these monoclonal antibodies reacted specifically with BHV-1 in an indirect fluorescent antibody test and immunoprecipitated a BHV-1 glycoprotein with molecular mass of 97 kilodaltons.     

Interaction of certain bovine immunoglobulins with complement in a single radial haemolysis system

Bovine IgG1, IgG2, and IgM initiated haemolysis by bovine complement. With guinea pig complement bovine IgG1 and IgM appeared effective, but bovine IgG2 was much less effective. Single radial haemolysis systems using guinea pig complement to measure bovine antibody are likely to detect predominantly IgG1 and IgM. However, in vivo IgG2 should activate bovine complement.     

Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin (Ig) M antibodies to bovine respiratory syncytial virus (BRSV) in cattle was developed. Monoclonal antibody to bovine IgM was used as the catching antibody. The IgM-ELISA was used, as well as a BRSV-specific IgG ELISA to determine the kinetics of IgM and IgG antibody responses to BRSV infections in cattle. High IgM and IgG antibody titers developed after naturally occurring or induced BRSV infection of calves (6 to 7 months old). Induced infection resulted in an IgM response that was first detectable at postinoculation day (PID) 11 reached a maximum at PID 13, and became undetectable again about PID 28. An IgG response also was detected by PID 11. However, a maximum response was not reached before PID 23, and titers remained high (until PID 80). In naturally occurring infection, IgM and IgG responses in calves were observed in the acute phase of epizootics of respiratory tract disease. Patterns of IgM and IgG response curves were similar to those observed in experimentally infected calves. The involvement of BRSV in an epizootic of respiratory tract disease in 8 calves (2 to 3 weeks old) was demonstrated by the detection of BRSV in several lung lavage samples. All calves had existing IgG antibodies to BRSV which were interpreted to be maternally derived. None of the calves responded with an increase in IgG antibody titer. However, a weak but distinct BRSV IgM antibody response occurred in 6 calves.(ABSTRACT TRUNCATED AT 250 WORDS)