Detection of antibodies to bovid herpesvirus 4 by ELISA

An enzyme linked immunosorbent assay for antibodies to bovid herpesvirus 4 was developed using antigen prepared by detergent lysis of infected cell cultures. The assay was used to study the immune responses of experimentally-immunised calves. The results correlated well with the indirect fluorescent antibody method. A viral neutralizing antibody response could not be demonstrated in the calves.     

Infection of sheep and goats with bovid herpesvirus 2

Sheep and goats were shown to be susceptible to experimental infection with bovid herpesvirus 2 (BHV2), administered by either the intradermal or intravenous route. Lesions developing in sheep following intradermal inoculation of virus were similar to those in cattle inoculated intradermally, whereas the lesions in goats resolved without ulceration or scabbing. Disseminated circumscribed skin lesions developed in sheep and goats given BHV2 by the intravenous route. These lesions resolved in four to eight days without significant effect on the skin. BHV2 was isolated from skin lesions of sheep, goats and cattle that were infected intravenously, from sheep and cattle infected intradermally and from the leucocytes of the three species following intravenous inoculation of virus. Latent infection of sheep and goats was demonstrated following corticosteroid treatment 60 days after infection.     

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.     

A comparison in calves of the antigenicity of three strains of bovid herpesvirus 2.

Three strains of bovid herpesvirus 2, viz. Allerton, bovine mammillitis and 69/1LO were used to infect calves intradermally. Twenty-eight days later the immunity of the calves was challenged by intravenous injection of a homologous or heterologous strain. Challenge control calves developed a fever (greater than 40 degrees C) lasting several days and widespread skin lesions which varied with the strain. Homologous challenge of the primary infection produced neither skin lesions nor febrile response, except in one calf in which fever was noted on one day. Heterologous challenge did not cause skin lesions but fever occurred in 8/12 calves. In particular Allerton virus failed to protect completely against heterologous challenge. Despite minor differences evident in these experiments, it is recommended that these isolates should be considered as strains of the same virus--bovid herpesvirus 2.     

First demonstration of bovine herpesvirus 2 infection among cattle by neutralization test in Japan

Seroepidemiological surveys were performed on neutralizing antibody to bovine herpesvirus 2 (BoHV-2) among cattle in Japan. A total of 1,819 sera were collected from cattle in 27 prefectures from 1997 to 1998. Antibodies were detected in only 18 sera collected from 4 prefectures. However, the most prevalent areas of the infection were found in two islands located in the subtropical zone. Additional 353 sera were collected in three including these islands in 1999-2001.The antibody-positive rates in the farms in these islands ranged from 10% to 81.1%. It was confirmed that BoHV-2 was prevalent in these areas. However, the infection seemed to be latent, because no diseases have been noticed. This is the first report showing the presence of BoHV-2 infection among cattle in Japan.     

Production of interferon by bovine peripheral blood monocytes infected with bovid herpesvirus 2

The production of interferon by bovine peripheral blood leukocytes infected with bovid herpesvirus 2 (BHV-2) was investigated in preparation for studying mechanisms of resistance to BHV-2. It was found that bovine peripheral blood monocytes produced high levels of interferon in response to BHV-2 inoculated at a multiplicity of 1. Virus-induced interferon was not stable at pH 2, was destroyed at 56 degrees C or by incubation with trypsin and was active against both vesicular stomatitis virus and BHV-2. Interferon of high specific activity was produced by incubating monocytes for 5 h with BHV-2 in serum-containing medium, replacing the inoculum with serum-free medium for an additional 16 h, and concentrating the serum-free medium by dialysis against dry polyethylene glycol. Interferon concentrations of 40,000 units per mg of protein were readily attained.     

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.     

The DNA of an IPV strain of bovid herpesvirus 1 in sacral ganglia during latency after intravaginal infection

Two calves were inoculated intravaginally with a strain of bovid herpesvirus type 1 (BHV-1, IBR/IPV) isolated from a cow with infectious pustular vulvovaginits (IPV). The animals were killed during a latent stage of infection as characterized by seroconversion, absence of virus shedding and recrudescence of virus shedding after dexamethsone treatment.IPV-virus DNA was detected in 9 out of 20 sacral ganglia of the 2 calves. Of the sections, 7.2% (n = 250) contained 1 cell with IPV-virus DNA, which was restricted to the nucleus of neurons. In agreement with findings on herpes simplex virus infections, the viral DNA of BHV-1 is harbored in the local sensory ganglia.Virological and serological implications of the latent IPV infection are discussed.     

Influence of isoprinosine on bovine herpesvirus type-1 infection in cattle

A study was conducted to determine the in vivo efficacy of isoprinosine (ISO) in calves infected with bovine herpesvirus type-1 (BHV-1). Calves were infected with BHV-1 on day 0 and received ISO daily for 14 days. Clinical signs of disease, shedding of BHV-1, lymphocyte proliferative responses to mitogens, interleukin-2 production, and alveolar macrophage bactericidal activity were monitored during the study. Rectal temperatures were increased (P less than 0.05) in BHV-1 and ISO-BHV-1 calves at days 3 to 7 postinfection (PI). Isoprinosine did not influence BHV-1 shedding in calves. Lymphocyte proliferative responses to phytohemagglutinin (PHA) were lower (P less than 0.01) in BHV-1 calves when compared to control or ISO calves at day 4 PI, but ISO did not ameliorate this effect. Interleukin-2 activity was greater (P less than 0.05) in ISO-BHV-1 calves on days 4 and 8 PI in PHA-stimulated lymphocytes and on day 8 PI in concanavalin A-stimulated lymphocytes when compared to control, ISO or BHV-1 calves. Isoprinosine treatment of BHV-1-infected calves tended to decrease alveolar macrophage bactericidal activity. These data suggest that ISO does not reverse BHV-1 suppression of lymphocyte proliferation, but may enhance IL-2 production in BHV-1 infected calves.     

Occurrence of ovine herpesvirus type-2 infection in sheep and cattle in Samsun Province, Turkey

July 2004, a cow with clinical signs of ovine herpesvirus type-2 infection which is known as sheep associated malignant catarrhal fever (SA-MCF) was reported in Samsun Province in Turkey. Blood samples were collected from the suspected cow, 10 sheep housed with it, and from 150 healthy sheep and 29 healthy cattle randomly selected from different places in Samsun Province. Nested polymerase chain reaction (n-PCR) was used to detect ovine herpesvirus type-2 (OvHV-2) DNA in the suspected cow and competitive- ELISA (c-ELISA) kits were used to detect antibodies against OvHV-2. The suspected cow was found to be n-PCR positive and c-ELISA negative. The serological results were as follows: All 10 (100%) of sheep housed with the suspected cow and 18 of 29 (62%) of the randomly selected cattle were found seropositive. All 150 randomly selected healthy sheep were seronegative. The overall percentage of seropositivity was 14.7% (28/190). OvHV-2 DNA was detected in the peripheral blood leucocyte (PBL) samples of the cow and of the 10 sheep housed with the suspected cow.     

Differential antigen recognition by serum antibodies from three bovid hosts of Mycobacterium bovis infection

Cattle, bison and buffaloes are susceptible to Mycobacterium bovis, the causative agent for bovine tuberculosis. Accurate and timely identification of infected animals is critical for improved management and control of disease in these species. Bovids develop humoral immune responses to M. bovis infection making serological tests attractive for tuberculosis screening. However, optimization and validation of antibody assays designed for various animal species require understanding of antigen recognition patterns in each target host. The objective of this study was to characterize serological reactivity profiles generated by cattle, American bison, and African buffaloes in tuberculosis. Serum samples from M. bovis-infected animals were tested for the presence of IgM and IgG antibodies to MPB70/MPB83 and CFP10/ESAT6 chimeric proteins using Dual-Path Platform technology. All three host species showed IgG responses of higher magnitude and frequency than IgM responses; further, IgM seroreactivity was limited to MPB70/MPB83, whereas IgG antibodies recognized both test antigens. In cattle, the IgM and IgG responses were elicited mainly by MPB70/MPB83, whereas bison and buffaloes showed similar IgG seroreactivity rates for MPB70/MPB83 and CFP10/ESAT6 antigens. The findings demonstrate distinct patterns of predominant antigen recognition by different bovid species in M. bovis infection.     

The virulence of British isolates of bovid herpesvirus 1 in relationship to viral genotype.

Six strains of bovid herpesvirus 1 isolated from British cases of infectious bovine rhinotracheitis (IBR) were inoculated intranasally into calves. The clinical, virological and serological responses were measured and comparisons made between virus strains. Calves infected with viruses of subtype 1 developed more severe clinical signs and excreted higher titres of virus than calves infected with subtype 2b strains. The findings could be related to the history of IBR in Great Britain.     

Detection of impaired T cell-mediated immune responses to herpesvirus (BHV-1) in cattle

Interleukin 2 (IL-2) functions in the regulation of cell-mediated immune responses both in vivo and in vitro. Therefore, IL-2 production has been studied in patients with immunological disorders to determine the level of the immune defect. However, the cause(s) of low responses to selected antigens by cells from clinically normal individuals has not been examined. The ability of cells from clinically normal individual cattle to produce and respond to IL-2 was investigated as a measure of specific cell-mediated immune response. Cells from the majority of animals (6/7 in a representative experiment) could produce IL-2 in response to mitogen or a specific antigen, Bovine Herpesvirus 1 (BHV-1). Proliferation of lymphocytes from the majority of low responding animals (2/3 and 4/4 in separate experiments) could be restored to a level similar to high responders by the addition of exogenous IL-2 after endogenous IL-2 depletion. IL-2 receptor expression was indirectly assessed by the ability of activated cells to absorb IL-2. One individual's cells were incapable of absorbing exogenous IL-2 and failed to proliferate indicating a lack of activation and expression of receptors. In addition, exogenous IL-2 was able to enhance proliferation of both high and low responders in the presence of endogenous IL-2. These results suggest that proliferation of bovine peripheral blood mononuclear cells is dependent on the presence of IL-2. In addition, IL-2 production can be used to measure specific cell-mediated immune responses.     

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).