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.     

Modelling the effect of surveillance programmes on spread of bovine herpesvirus 1 between certified cattle herds

For the eradication of an infectious agent, like bovine herpesvirus 1 (BHV-1), surveillance and certification can be used to reduce the transmission between herds. The goal of surveillance is that a certified herd that becomes infected is detected timely so that infection of several other certified herds is prevented. What counts is whether the reproduction ratio R, i.e. the average number of certified herds infected by one infected certified herd can be kept below 1. To support policy makers in making decisions about the minimal demands for a surveillance programme in an eradication campaign of BHV-1 in cattle, two mathematical models were investigated. With these models, the basic reproduction ratio between herds was calculated. The surveillance programmes were characterised with sample size, sampling frequency, test sensitivity, herd size, vaccination status, and contacts between herds. When R between herds is below 1, then the surveillance programme is sufficiently good to prevent spread of infection, provided that R is estimated well. In the model based on bulk milk testing sample size was replaced by a threshold at which bulk milk can be found positive. The R between herds was mainly influenced by the vaccination status, sampling frequency, and contacts between herds. Herd size moderately affected the outcome. Test sensitivity and sample size, however, were of minor importance. If herds of 50 cows became free of BHV-1 without vaccination, then spread of infection between herds might be prevented when animals within herds are sampled once a year (milk or blood samples). This frequency needs to be intensified, being twice a year, for larger herds and/or herds with extensive contacts with other herds. When bulk milk is sampled instead, sampling should be done at least every 5 months and more intensively, being each month, with larger herd sizes and more contacts between herds.     

Effect of viral dose on experimental pneumonia caused by aerosol exposure of calves to bovine herpesvirus 1 and Pasteurella haemolytica.

The effect of various aerosol doses of bovine herpesvirus 1, followed four days later by aerosol exposure to a constant level of Pasteurella haemolytica, was studied in 16 crossbred Hereford range calves. A Collision nebulizer was used to generate aerosols from virus suspensions with concentrations of 10(8.2) (high), 10(5.2) (moderate) or 10(2.2) (low) TCID50/mL. The bacterial suspension contained 10(7) colony forming units/mL. Control calves exposed only to P. haemolytica developed no pulmonary lesions. Calves in the low, moderate and high virus exposure groups developed lobular areas of atelectasis; in addition, one calf in the moderate and all four in the high virus exposure group developed fibrinous pneumonia. One of the latter calves died. The 50% effective dose for fibrinous pneumonia under these experimental conditions was 10(6.0) TCID50 bovine herpesvirus 1/mL of suspension in the nebulizer reservoir, and approximately 10(4.0) infectious units inhaled per calf.     

Prevalence of bovine herpesvirus 1 (BHV-1) infection in Hungarian cattle herds.

Hungarian cattle herds were surveyed for bovine herpesvirus 1 (BHV-1) infection by ELISA of milk and serum samples. In 1993, 75% of the large cattle herds (consisting of more than 50 cattle) and all small herds (small-scale producers' stocks), while in 1997 90% of the small herds were included in the survey. In the case of large herds, 79.3% of the herds and 64.1% of the samples tested were found to be positive. Of the small herds, 13.5% and 15.7% tested positive in 1993 and 1997, respectively. The majority of large herds were Holstein-Friesian dairy stocks. Small herds with an infection rate markedly exceeding the average were found in those counties where the small herds had been in close contact with the large-scale farms, or where new herds were established by using animals of uncontrolled infectious bovine rhinotracheitis (IBR) status originating from large farms. Attention is called to the importance of maintaining the IBR-free status of small herds that constitute one-third of the Hungarian cattle population.     

Effect of repeated intradermal injections of bovine herpesvirus type 1 antigen on seronegative cattle.

Forty-three cattle seronegative to bovine herpesvirus-1 (BHV-1) were given from one to five intradermal injections of BHV-1 inactivated antigen at four-week intervals. This delayed hypersensitivity test was assessed by the increase in skin thickness. The activity of the antigen was assessed in five animals which had a previous natural BHV-1 infection with clinical signs and seroconversion. Anti-BHV-1 antibodies were detected by seroneutralisation and an enzyme-linked immunoassay. Only one animal showed a significant but slight increase in skin thickness after the first test, but it was negative after a second test. The animals remained seronegative after the first test. Seroconversion was identified in 11 of the 43 animals (25 per cent) submitted to repeated delayed hypersensitivity tests. Five of 37 animals seroconverted after only two tests. The serological response was transient in seven of 11 seroconverted calves. Repeated hypersensitivity tests were therefore able to induce a serological response in seronegative calves but the response was weak and often transient. The test must therefore be applied cautiously to seronegative animals.     

Frequency of occurrence of viruses associated with respiratory tract disease of cattle in Oklahoma: serologic survey for bovine herpesvirus DN599.

Serum samples from 351 Oklahoma cattle were tested for antibodies to DN599 virus by the indirect fluorescent antibody test. Seven cattle (approximately 2%) were seropositive for this virus.     

Serological titers to bovine herpesvirus 1, bovine viral diarrhea virus, parainfluenza 3 virus, bovine respiratory syncytial virus and Pasteurella haemolytica in feedlot calves with respiratory disease: associations with bacteriological and pulmonary cytological variables.

Acute and convalescent serum samples were taken from 59 calves with signs of respiratory disease (cases) and 60 clinically normal animals (controls) during their first month in the feedlot. Sera were analyzed for antibodies to bovine parainfluenza 3 (PI3) virus by hemagglutination inhibition, to bovine viral diarrhea (BVD) virus, bovine respiratory syncytial (BRS) virus and bovine herpesvirus 1 (BHV1) by virus neutralization, and to Pasteurella haemolytica by indirect agglutination (PhIA) and cytotoxin neutralization (PhCN) tests. There was minimal evidence of serological activity to BHV1. Serological activity to the other agents occurred commonly and the prevalence of acute titers and their mean values was similar in case and control groups. Mean convalescent PI3 and P. haemolytica (PhIA) titers were higher in controls than cases (p < 0.01) but, otherwise, convalescent titers did not differ between groups. The incidence of seroconversion was similar in both groups for all agents except for PI3 virus which was more frequent in controls than cases (p < 0.0001). There was a positive association between PhIA and CN seroconversion and isolation of P. haemolytica from bronchoalveolar lavage (BAL) fluid (p < 0.1). The measure of agreement (kappa) between seroconversion with the P. haemolytica PhIA and PhCN tests was 0.51. Bacteriological and cytological evaluations of the respiratory tract were made using BAL. No associations were evident between serological titers and pulmonary cytology. A multivariate logistic analysis was used to evaluate associations between disease status and serological, bacteriological and cytological data. Cases were positively associated with the presence of neutrophils and Pasteurella multocida in BAL fluid and negatively associated with PI3 virus and PhIA seroconversion.     

Development of a polymerase chain reaction and restriction typing assay for the diagnosis of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.     

Cross-reactions of bovine herpesvirus 1 antigens with those of other cattle herpesviruses

Bovine embryonic kidney cells were infected with bovine herpesviruses (BHV1, 2, or 3), suid herpesvirus 1 (SHV1), or were sham-inoculated. When cytopathic effect was apparent, the cells were solubilized using Triton X-100 detergent. Resulting antigen preparations were tested by 2-dimensional immunoelectrophoresis using bovine fetal serum and antisera directed against BHV1, BHV2, BHV3, SHV1 or a restricted spectrum of BHV1 antigens. Interaction of BHV1 antiserum with BHV1 antigen preparations resulted in 11 precipitation arcs. The same antiserum produced 3 arcs with BHV2, none with BHV3, and 5 with SHV1. The interaction of BHV1 antigen preparations with BHV2, BHV3, or SHV1 antisera failed to produce demonstrable arcs. However, when heterologous antigen or antibody preparations were added to BHV1 homologous 2-dimensional immunoelectrophoresis tests, all 11 BVH1 arcs were modified by BHV1, 2 by BHV2, 4 by BHV3 and 4 by SHV1 preparations. Two antigens were common to the 4 herpesviruses. Antigen preparations were tested for their ability to inhibit virus neutralization by BHV1 antiserum; only the BHV1 preparation was active. Sera were tested for BHV1 neutralizing activity; only BHV1 antiserum and a serum specific for a restricted spectrum of BHV1 antigens were active. A glycoprotein antigen associated with BHV1 neutralization was identified which may be important in the protection of animals against disease.     

Effect of in ovo vaccine delivery route on herpesvirus of turkeys/SB-1 efficacy and viremia

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.     

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.     

Quantification of the contact structure in a feral cattle population and its hypothetical effect on the transmission of bovine herpesvirus 1

The organisation of animal populations in social groupings may play a crucial role in the transmission of any infectious disease that requires close contact. The objective of this study was to quantify the contact structure of part of the Heck cattle population in a Dutch nature reserve and its hypothetical effect on the transmission of bovine herpesvirus 1 (BHV1). The contact structure was quantified by observing the number of different animals with whom contact was made (i.e. the number of contactees) within a fixed time period. Two types of behaviour sampling methods, namely focal sampling and scan sampling were used to observe the contact structure. In this study only those contacts between individuals were observed that were assumed to be a proxy measure of an at-risk event for BHV1-infection. Two reproduction ratios (R), i.e. the average number of new cases caused by a typical infected individual, were estimated, one for the observed contact structure and another for a random mixing contact structure. The two reproduction ratios were then compared to study the hypothetical effect on BHV1 transmission.

The overall number of contactees was highest during summer and lowest during winter-spring. The contact structure of the homogeneous population did differ significantly from a random mixing contact structure, resulting in that the variation in the number of contactees was higher than under random mixing. Bulls, young bulls and cows had the highest number of contactees during, respectively, summer, autumn and winter-spring. From the analysis of the contingency tables it was clear that contacts between animal types did not occur at random during summer and autumn. For example, during summer more contacts than expected occurred between bulls and cows. This heterogeneity at animal type level was taken into account in the calculation for R, which resulted for the observed contact structure in higher estimates for R than for the homogeneous population.

When looking at heterogeneity at individual level it was found that during summer almost all individuals were observed together direct or indirect in the same group except for certain bull groups. During autumn and winter-spring almost all individuals were seen together in the same group when considering a long contact period of 14 days but the groups were fallen apart in smaller groups and solitary individuals for a short contact period of 5 days.

It could be concluded that based on the observed contact structure transmission would be favoured most during summer.     

Immune response of cattle to antigens obtained from bovine herpesvirus 1-infected tissue culture.

The ability of two antigens, termed EV and CM, derived from bovine herpesvirus 1 infected cultures to produce serum-virus neutralizing antibodies has been studied in cattle. Both EV and CM when inoculated with adjuvant induced significant increases in serum-virus neutralizing antibody titers. Control groups inoculated in a similar manner failed to induce significant increases in serum-virus neutralizing antibody. Some of the animals were vaccinated, then were bred, challenged with a virulent strain of bovine herpesvirus 1 and held until calving was completed. During this 18-month period titers declined slowly in the vaccinated animals. Proportionally there were fewer live calves born to the control cattle than to the CM vaccinated group but reduction was not large enough to conclude that this vaccine had protected the cattle against the abortigenic activity of bovine herpesvirus 1. Further challenge studies should be made to determine whether the administration of these antigens can prevent the subsequent onset of the clinical signs associated with bovine herpesvirus 1.     

Biosecurity and bovine respiratory disease.

Although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. Because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. Several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. Various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease.