Pathogens at the livestock-wildlife interface in Western Alberta: does transmission route matter?

In southwestern Alberta, interactions between beef cattle and free-ranging elk (Cervus elaphus) may provide opportunities for pathogen transmission. To assess the importance of the transmission route on the potential for interspecies transmission, we conducted a cross-sectional study on four endemic livestock pathogens with three different transmission routes: Bovine Viral Diarrhea Virus and Bovine Herpesvirus 1 (predominantly direct transmission), Mycobacterium avium subsp. paratuberculosis (MAP) (indirect fecal-oral transmission), Neospora caninum (indirect transmission with definitive host). We assessed the occurrence of these pathogens in 28 cow-calf operations exposed or non-exposed to elk, and in 10 elk herds exposed or not to cattle. We characterized the effect of species commingling as a risk factor of pathogen exposure and documented the perceived risk of pathogen transmission at this wildlife-livestock interface in the rural community. Herpesviruses found in elk were elk-specific gamma-herpesviruses unrelated to cattle viruses. Pestivirus exposure in elk could not be ascertained to be of livestock origin. Evidence of MAP circulation was found in both elk and cattle, but there was no statistical effect of the species commingling. Finally, N. caninum was more frequently detected in elk exposed to cattle and this association was still significant after adjustment for herd and sampling year clustering, and individual elk age and sex. Only indirectly transmitted pathogens co-occurred in cattle and elk, indicating the potential importance of the transmission route in assessing the risk of pathogen transmission in multi-species grazing systems.

The effect of the mode of sampling on BHV-1 detection in infected cattle by dot-blot hybridization.

The BHV-1 genome in nasal swabs and washings was detected by dot-blot hybridization using the 32P-pUR-1 probe (1.8 kb EcoRI-HindIII random fragment of BHV-1 DNA ligated into the pUC-9 plasmid) as early as on day 1 after the experimental infection of cattle. In dependence on the sampling method, differences were observed in the maximum of hybridization signals. During nasal swab analyses maximum amounts of BHV-1 differed in the individual samples (day 1-3). Hybridization signals obtained at the analysis of BHV-1 DNA nasal washings did not vary but showed a continuous maximum on day 2 after infection. Nasal washings proved to be more advantageous for detection of the BHV-1 genome by the hybridization technique.

A comparison of the virulence of three strains of infectious bovine rhinotracheitis virus.

The virulence of three strains of infectious bovine rhinotracheitis (IBR) virus was compared in six-month-old Ayrshire-cross calves. The strains were an isolate from a recent severe outbreak of IBR in Scotland (Strichen strain), the prototype British strain (Oxford strain) and a North American isolate (Colorado strain). The Colorado and Strichen strains produced the characteristic clinical signs and pathological lesions of severe IBR three to four days post infection (p.i.). The Strichen strain was slightly more virulent, possibly as a result of its having been passaged fewer times in tissue culture. In contrast, the Oxford strain produced a mild clinical response with minimal pathological lesions. Virus was recovered from nasal swabs for a longer-period from the calves infected with the Strichen strain (up to 13 days p.i.) and Colorado strain (up to 12 days p.i.) than from the animals infected with the Oxford strain (up to 10 days p.i.). These findings support the suggestion that the recent epidemic of severe IBR in Britain had resulted from the importation of a "new" strain of virus.

Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts.

Bovine herpesvirus 1 (BHV1) and caprine herpesvirus 1 (CapHV1) are useful models to study virus-host interactions, as well as pathogenicity and latency, when comparing the outcome of infection in the natural and the foreign hosts. Molecular seroepidemiological analyses revealed that cross-reacting antibodies were mainly induced by glycoprotein gI (gB analogue), by the major capsid protein and by nonstructural proteins, whereas the most virus-specific antibodies were elicited by glycoproteins gIII and gIV. These glycoproteins, especially gIII (gC analogue), might therefore play an important role in the virus-host-interactions. As a basis for further studies, we re-evaluated observations concerning experimental infections with BHV1 and CapHV1 in the natural and the foreign hosts. All parameters indicated that both viruses were able to infect either host, but that the pathogenicity was restricted to the natural host. Latent virus could be reactivated exclusively from cows infected with BHV1. It was possible neither to reactivate BHV1 from goats, nor to reactivate CapHV1 from either species. The experiments indicated that the outcome of infection in the natural and the foreign host is dependent on host and viral factors, whereby gIII is only one important virus component involved. Further investigations in the host and host cell range of BHV1 and CapHV1 will help to clarify the role of factors responsible for virus-host-interactions.

Variability occurs in the inverted repeat region of genomic DNA from bovine herpesvirus 1 respiratory, genital and bovine herpesvirus 5 encephalitic isolates.

Restriction fragment length polymorphisms (RFLPs) were detected within BHV1.1, BHV1.2, and BHV5 genomes using the restriction enzyme PstI. The genomic areas of these changes has not been previously reported. Using Southern blot hybridization with DNA probes representing the entire genome of BHV1.1, areas of genomic variation were located for a respiratory isolate (BHV1.1), four vaccine isolates (BHV1.1), a genital isolate (BHV1.2), and two encephalitic isolates (BHV5). The most frequently observed RFLPs of BHV1.1 and BHV1.2 occurred within the internal repeat region and the left terminus of the unique long region. When two separate isolates of the encephalitic BHV5 were compared, RFLPs were detected in the internal and right terminal repeat regions. These are the regions of each genome from which immediate early genes are transcribed. No genomic variation was observed throughout the unique long and unique short regions for all BHV1 and 5 isolates examined.

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 vulvovaginitis (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 dexamethasone 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.

Excretion and reexcretion of thermosensitive and wild-type strains of infectious bovine rhinotracheitis virus after co-infection or two successive infections.

Twelve cattle were divided into 2 groups. The first was intranasally co-infected with 2 strains of infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1): the thermosensitive vaccine strain IBR/ts RLB106 and a Belgian field isolate IBR/Cu5. Reactivation of BHV 1 was induced by dexamethasone treatment 2 months later and again 5 months later for 3 animals that only reexcreted small quantities of virus during the first dexamethasone treatment. The second group was intranasally infected with IBR/Cu5. Two months later, an attempt to reinfect this group with IBR/ts RLB106 failed. Four months after the primary infection, these cattle were treated with dexamethasone. Except after reinfection and at the beginning or the end of the (re)excretion periods, excreted and reexcreted viruses replicated at 35, 37 and 40 degrees C, indicating the presence of the wild-type virus. Only one isolate, out of 116 cloned from the nasal exudates collected during the excretion and reexcretion periods, expressed the thermosensitive phenotype. This isolate was characterized by its mean plaque size as the IBR/ts RLB106 strain. The epizootiological significance of these findings is discussed, with emphasis on the weak spreading capacity of the ts vaccine strain and the possibility of emergence of recombinant viruses.

Reactivation of infectious bovine rhinotracheitis virus by transport.

Transport was studied as a cause of reactivation of infectious bovine rhinotracheitis virus (Bovine herpesvirus-1; BHV-1) in heifers vaccinated 2-6 months before transport, using a double dose of the thermosensitive (ts) vaccine strain (Tracherine). Eight out of 19 animals showed ts strain re-excretion over a period of 1-3 days, beginning, in 5 out of the 8 heifers, the day after transport. In 14 other heifers, only sera were examined by sero-neutralisation: only 1 out of these 14 animals showed a rise in BHV-1 neutralising antibodies. Transport can therefore be considered as a stimulus of BHV-1 reactivation.

Comparative studies of strains of infectious bovine rhinotracheitis virus isolated from latently infected calves.

Three strains (479 C, 778 TL, 982 LE) of infectious bovine rhinotracheitis (IBR) virus isolated from latently infected calves were compared with the prototype strain of IBR virus (LA strain) in studies which included restriction endonuclease analysis, experimental infection, and reciprocal cross protection tests in cattle. From the restriction endonuclease analysis it appeared that the 3 "latent" viruses were derived from the same isolate, and that it differed slightly from the LA strain. However, latency does not seem to have affected the pathogenicity or the immunogenicity of the virus. This is demonstrated by the identical clinical and virologic response of calves subjected to experimental infection with the various strains under study, and by the finding that when the LA strain and a "latent" strain (982 LE) were tested in cross protection tests in cattle, they proved to be mutually protective.

Demonstration of infectious bovine rhinotracheitis virus antigen in paraffin sections.

Nine pregnant heifers were inoculated intravenously with infectious bovine rhinotracheitis virus (IBRV) in the sixth month of pregnancy. Tissues were collected from the fetus of a heifer killed 13 days postinoculation (PI), from fetuses of 6 heifers that aborted 16-27 days PI, and from mummified fetuses of 2 heifers that aborted 53 and 85 days PI, respectively. Control tissues were obtained from the fetus of a non-inoculated heifer that was killed in the seventh month of gestation. Tissues were fixed in 10% formalin, embedded in paraffin, and examined for viral antigen by immunohistochemistry, using biotinylated second antibody and alkaline phosphatase-labeled avidin-biotin complex. Antigen was detected in at least 1 tissue from the fetus of each inoculated heifer. Positive tissues included lung, liver, spleen, kidney, adrenal, and placenta. In several fetuses, antigen was identified in tissues from which virus was not isolated in cell culture. This appeared to occur when tissues had only a few small foci of infection or when tissues were severely autolyzed. The observation of viral antigen in tissues from mummified fetuses indicates that this technique may be useful in diagnostic laboratories to detect IBRV infection in tissues that are not suitable for virus isolation or for examination by the cryostat tissue section-fluorescent antibody technique.

Use of in situ hybridization with a biotinylated probe for the detection of bovine herpesvirus-1 in aborted fetal tissue.

Forty-five cases of bovine abortion were examined using in situ hybridization (ISH) with a biotinylated DNA probe specific for bovine herpesvirus-1 (BHV-1). Of the 45 cases, 16 were diagnosed as due to BHV-1, 15 were determined to be due to other causes, and 14 were of undetermined etiology. Direct comparisons between ISH and an immunoperoxidase (IP) test specific for BHV-1 were performed on formalin-fixed, paraffin-embedded tissue sections of lung, liver, kidney, spleen, thymus, and placenta; fluorescent antibody tests for BHV-1 and virus isolation were performed on fresh lung and liver. In comparison to these routine BHV-1 detection techniques, ISH had an overall sensitivity of 88.2% and a specificity of 89.3% in detecting BHV-1 in aborted fetuses. Immunoperoxidase was more sensitive than ISH with tissue sections from lung (87.5% vs. 69%), liver (92% vs. 17%), spleen, and placenta; results of the tests on tissue sections from kidney were concordant. Liver sections presented special problems in that nonspecific reactions were frequently observed with hybridization. With thymus sections, the rate of detection was higher by hybridization than by IP, but the specificity of some of these reactions could not be confirmed.

Reactivation and shedding of bovine herpesvirus 1 following Dictyocaulus viviparus infection.

Three groups of 4 bullocks which had recovered from infectious bovine rhinotracheitis (IBR) were infected 5 months later with Dictyocaulus viviparus larvae. Bovine herpesvirus 1 was recovered from days 7 to 21 post-infection from the nasal secretions of the group given 50 larvae per kilogram and on one occasion from those given 1000 larvae per animal (less than 5 L3 per kg). Virus was not isolated from the animals given 1000 irradiated larvae. Typical clinical signs and lesions of IBR developed in the group from which the virus was isolated regularly.

Experimental infectious bovine rhinotracheitis: comparison of four antigen detection methods.

The duration of detectable viral antigen in nasal secretions was studied by four methods in calves experimentally infected with infectious bovine rhinotracheitis. Virus isolation in cell culture was the most sensitive and was successful for up to 11 days after inoculation. Three direct rapid antigen detection systems (immunofluorescence, immunoperoxidase and enzyme-linked immunosorbent assay [ELISA]) were all successful during the pyrexic phase, but less useful in the later stages of the disease. Virus isolation and ELISA remained positive in strongly positive samples stored for a week at room temperature.

Bovine herpesvirus-1 and parainfluenza-3 virus interactions: clinical and immunological response in calves.

Calves infected with bovine herpesvirus-1 (BHV-1) or both BHV-1 and parainfluenza-3 virus (PIV-3) developed clinical signs including fever, cough, and nasal and ocular discharges. Animals infected with both viruses appeared more depressed and showed higher rectal temperature, while calves inoculated with PIV-3 alone had a very mild clinical disease. Both BHV-1 and PIV-3 were recovered from nasal secretions up to six to eight days postinoculation. However, the virus titers were lower in calves with mixed infection. An increase in serum antibodies to both BHV-1 and PIV-3 was detected by serum neutralization and enzyme-linked immunosorbent assay. Antibody responses were delayed and significantly lower in calves given mixed infection than in calves infected with a single virus. Interleukin-2 activity in cultures of lymphocytes from BHV-1 and BHV-1 plus PIV-3 infected calves was higher compared to control calves.

Bovine herpesvirus-1 and Pasteurella haemolytica aerobiology in experimentally infected calves.

Eight calves (2 calves in each of 4 groups) were exposed to an aerosol of bovine herpesvirus-1 (BHV-1) and 4 days later to an aerosol of Pasteurella haemolytica. Samples of tracheal and exhaled air were taken simultaneously beginning 1 day before viral exposure and once a day up to 3 to 4 days after the bacterial exposure. Samples were also taken during the period of aerosol exposure. Only 0.04% to 0.42% of P haemolytica-carrying droplets of the bacterial aerosol passed beyond the cranial part of the respiratory tract to the trachea. Nevertheless, numbers of bacteria as few as 1 bacterium/L of tracheal air were sufficient to produce fatal disease in the lungs of BHV-1-infected calves. In 1 of 4 groups, BHV-1 was isolated from most daily samples of exhaled and tracheal air. Pasteurella haemolytica was isolated 7 times more frequently from air when calves were kept at 1 C than when calves were kept at 23 C. The number of P haemolytica-carrying droplets in exhaled air was low (less than 1/L of air); however, samples obtained during the time that calves were coughing contained up to 10 P haemolytica-carrying droplets/L of air. It was learned that the cranial part of the respiratory tract serves as an efficient filter on inhalation and exhalation, but this filter is deficient in the animal when coughing occurs. This process expels infective droplets of size suitable for inhalation by other cattle in close proximity.

Pasteurella haemolytica serotype 1 colonization of the nasal passages of virus-infected calves.

Nasal passages of calves with a virus-induced respiratory tract disease became colonized by Pasteurella haemolytica serotype 1 after they were inoculated intranasally with P haemolytica. Inoculation with infectious bovine rhinotracheitis virus caused a more severe clinical illness and resulted in a greater degree of colonization with P haemolytica than developed after inoculation with parainfluenza-3 virus. Nasal passages of parainfluenza-3 virus-inoculated calves were colonized to a greater degree with P haemolytica than were those of healthy, nonstressed calves. Calves were susceptible to P haemolytica colonization during or shortly after virus-induced illness, even though they had been previously exposed to P haemolytica and had serum antibody and nasal secretion antibody to P haemolytica.

Combined effects of fasting and diet on interferon production and virus replication in calves infected with a vaccine strain of infectious bovine rhinotracheitis virus.

A study was undertaken to investigate the combined effects of fasting and different diets on interferon (IFN) production and virus replication measured in nasal secretions of calves inoculated with a vaccine strain of infectious bovine rhinotracheitis virus. Four groups of calves were inoculated intranasally with infectious bovine rhinotracheitis virus. Two groups were inoculated 24 hours after onset of a 3-day fast; upon refeeding, 1 group was fed a maintenance diet (M diet) of hay, and the other was fed a higher energy diet (HE diet) of hay and concentrate. Nonfasted control groups were fed the M diet or the HE diet. Overall IFN production was highest (P less than 0.01) in nonfasted calves fed the M diet throughout the study and lowest in nonfasted calves fed the HE diet. Fasted calves refed the HE diet produced consistently and significantly more IFN than did nonfasted calves fed this diet. Fasted calves refed the M diet, however, produced significantly less IFN, compared with control calves fed the M diet throughout the study. Overall mean virus excretion was similar in all groups; therefore, the amount of virus replication per se did not account for the differences in IFN production, nor did greater IFN production result in less virus excretion. Serum cortisol concentrations and immune responses were not significantly affected by fasting or diet.

Clearance and shedding of infectious bovine rhinotracheitis virus from the nasal mucosa of immune and nonimmune calves.

Infectious bovine rhinotracheitis virus was rapidly cleared from the nasal mucosa of calves after intranasal aerosol exposure. Nonimmune calves (experiment 1) cleared 10(9) plaque-forming units (PFU) of virus from the nasal mucosa in less than 4 hours and 10(6) PFU of virus in 1 hour. An eclipse phase followed the clearance of viral inoculum. Replicating virus was first detected at 9 hours. Viral titers increased stepwise until maximum was attained on postinoculation day 4. Virus persisted in the nasal mucus until day 12. Clinical signs of disease corresponded with the shedding of virus. In contrast to nonimmune calves, immune calves (experiment 2; same calves as in experiment 1, but 30 days after initial exposure) cleared 10(9) PFU of virus in 1 hour and 10(6) PFU of virus in less than 5 minutes. An abortive reinfection occurred after exposure of immune calves with 10(9) PFU of virus. Virus was first detected in these calves at 14 hours after exposure and was not detected beyond 24 hours after inoculation. Immune calves given 10(6) PFU of virus did not shed virus after clearance of inoculum. Clinical signs of infection were not observed in immune calves after viral challenge exposure. The date indicated that there was no detectable residual virus beyond 3 hours after the exposure.

Chemotherapeutic value of 2-deoxy-D-glucose in infectious bovine rhinotracheitis viral infection in calves.

Daily injection of 2-deoxy-D-glucose (2-dG) had no protective effect against respiratory tract infection in calves caused by infectious bovine rhinotracheitis virus. It also did not reduce the severity of this infection. Ocular instillation of the drug, however, markedly reduced the severity of viral-induced conjunctivitis and keratoconjunctivitis. The drug was effective when given at the time of ocular infection or after clinical conjunctivitis developed.

Effect of infectious bovine rhinotracheitis virus infection of calves on cell populations recovered by lung lavage.

Calves were challenge exposed with infectious bovine rhinotracheitis (IBR) virus, and experiments were carried out to determine the presence of virus in the lungs, virus or viral antigen in alveolar macrophages, and alterations in immune functions of alveolar macrophages. In experiment 1, calves were challenge exposed intranasally with IBR virus. Although clinical signs of IBR occurred in all challenge-exposed calves, there was minimal evidence of virus or viral antigen in cells lavaged from their lungs, and macrophage Fc- and complement-receptor activities, phagocytic activity, and ability to mediate antibody-dependent cell cytotoxicity were unaltered. In experiment 2, calves were challenge exposed intranasally or by aerosol with IBR virus strains Colorado-1 and 108, and samples were collected 4 and 6 days after challenge exposure. Virus was isolated from the lungs, and pathologic lesions of greater severity occurred in those calves challenge exposed by aerosol. Less than 0.1% of lavaged cells from challenge-exposed calves produced infectious centers on susceptible cell monolayers. In 1 sample of lavaged cells, approximately 5% of the cells, mainly macrophages, had viral antigen in the cytoplasm, as detected by immunofluorescence. Because of the small proportion of macrophages that appeared to become infected after challenge exposure of calves with IBR virus, it is believed that the effect of IBR virus in predisposing calves to pneumonic pasteurellosis is an indirect, rather than a direct, manifestation of viral infection of macrophages.