Transmission of Ovine Herpesvirus 2 in Lambs

Abstract

The pattern of acquisition of ovine herpesvirus 2 (OHV-2) infection in lambs was examined by a competitive-inhibition enzyme-linked immunosorbent assay and PCR. Newborn lambs (n = 118) did not exhibit antibody at birth. Viral DNA in peripheral blood leukocytes was detected in only 3% (n = 77) of newborn lambs before suckling. After nursing, viral DNA was sporadically present in about 10 to 20% of lambs until about 3 months of age. Thereafter, strong DNA signals began to appear in increasing numbers of lambs, reaching 100% by 5.5 months of age. Viral DNA in nasal secretions began to be detectable in about 30% of lambs at 5.5 months of age, achieved significant levels in 88% of lambs by 7.5 months of age, and then declined. The kinetics of the humoral immune response in lambs paralleled those of viral DNA in nasal secretions but did not parallel its presence in blood leukocytes. In the experiment to define the time of infection of OHV-2 in lambs, all five lambs separated from the flock at 2.5 months of age remained uninfected until the termination of the experiment at 1 year of age. In contrast, lambs weaned at 2.5 months of age and returned to the flock had become infected at 3.5 months of age. Weaning and separation from the flock at 3.5 months of age did not prevent infection. The study showed that OHV-2 infection does not commonly occur in perinatal lambs and that OHV-2-free sheep can be established by separation of lambs at the proper time, which has important implications for potential control measures.

Classical malignant catarrhal fever (MCF) is a serious and often lethal infection of many species of the families of Bovidae and Cervidae (6, 20, 26). Its hallmarks are a syndrome of widespread lymphoproliferation, vasculitis, and inflammatory lesions affecting many tissues. Of several closely related viruses capable of inducing the syndrome of MCF, only one has been relatively well characterized. This one, classified as a gammaherpesvirus and termed alcelaphine herpesvirus 1 (AHV-1) (23), is endemic in wildebeests, to which it is well adapted, causing virtually no clinical disease (18). When certain other, less well adapted ruminant species become infected, the clinical disease syndrome develops (19). Where free-ranging wildebeests are not present, which is in most of the world outside Africa, other carriers serve as the source of MCF virus. The identity of the other source(s) of virus has long been elusive, but a large body of evidence has accumulated over the past 50 years to implicate domestic sheep (21). Classical experiments showed that mixing clinically susceptible species with sheep could lead to cases of MCF (cited by Plowright [20]). A high percentage of domestic sheep have antibodies that react with AHV-1 antigens (5, 9, 13, 27). The recent detection of DNA sequences similar to those of AHV-1 from lymphoblastoid cell lines derived from cattle or deer with clinical sheep-associated MCF (SA-MCF) (1, 3) and normal sheep (1, 10) further strengthens the concept that sheep indeed carry the causative agent for SA-MCF. However, many attempts to isolate the virus from sheep or from animals with clinical SA-MCF have failed. Nevertheless, the agent has been designated ovine herpesvirus 2 (OHV-2) on the basis of its antigenic and base sequence relatedness to AHV-1 (25).

Information about transmission of OHV-2 among sheep is relatively sparse. The occurrence of SA-MCF in cattle or other ruminant species is often associated with lambing (4, 22, 23), which parallels the association of wildebeest-associated MCF with calving wildebeests (14). The similarity in seasonal occurrence of disease between SA-MCF and wildebeest-associated MCF led to speculation that lambs, like wildebeest calves, serve as an important source for transmission. However, recent studies have suggested that the pattern of OHV-2 transmission between sheep might differ from that of AHV-1 transmission between wildebeests (10). In this study, competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA) for specific antibody to MCF virus (9) and PCR for an OHV-2 DNA fragment (1) were employed to investigate the natural transmission of OHV-2 in lambs.

MATERIALS AND METHODS

Animals and experimental designs.

The study utilized sheep flocks at two different locations: the University of Wyoming, Laramie, and the USDA Sheep Experiment Station, Dubois, Idaho. In Wyoming (experiment 1), 12 lambs were born to OHV-2-infected ewes in February of 1994. The lambs were raised under normal flock conditions, weaned at 3 months of age, and separated immediately from the infected ewes. The lambs were then housed in an enclosed facility which was at least 100 yd away from any other sheep. Fifteen lambs born to OHV-2-infected ewes in March of 1994 were used as a control group in Dubois, Idaho. These lambs were weaned at 3 months of age and then returned to the flock and raised under normal husbandry conditions. Serum samples were taken regularly from both groups of lambs for seroassay.

An experiment designed to define the time point when infection occurs in lambs was performed the following year at Dubois, Idaho (experiment 2). Two groups of 10 lambs comprised of five sets of twins from OHV-2-infected ewes were raised under normal conditions and weaned at either of two intervals: 2.5 and 3.5 months of age. At weaning time (2.5 or 3.5 months of age), one member of each twin pair (n = 5) was immediately placed into biosafety level 2 isolation facilities. The other half of each group (comprised of the other member of each of the five pairs) was returned to the flock after a 10- to 14-day weaning period. The two different weaning-age groups in isolation were maintained separately from one another to avoid cross-infection.

To define the appearance of viral DNA in nasal secretions, peripheral blood leukocytes (PBLs), and serum antibody in lambs under normal husbandry conditions, 43 lambs from OHV-2-infected ewes were sampled for the first 3 months and 17 lambs were removed from this flock at weaning time, leaving 26 lambs. These were sampled through the end of the experiment. Both blood samples and nasal swabs were collected from each monthly until termination of the experiment.

Fetal examination.

To assess the possibility of transplacental infection, four fetuses were obtained surgically from OHV-2-infected ewes 5 days prior to parturition. Anticoagulant-treated blood for plasma and leukocyte sampling was collected in EDTA tubes, and lymph node samples were collected from each fetus.

Sample collection and preparation.

A total of 118 serum samples and 77 PBL samples were obtained from presuckling lambs, and 70 serum samples were obtained from lambs after nursing in Wyoming and Idaho. The samples collected from the lambs in the above-described experiments were also included in the total numbers. Nasal secretions and EDTA-anticoagulant-treated peripheral blood for leukocyte and plasma sampling were collected at each sampling interval. Nasal secretions were obtained by inserting two cotton-tipped, wooden-handled swabs into one nostril to a depth of approximately 3 in., withdrawing them after 2 min, and placing them in a tube containing 1.0 ml of cold phosphate-buffered saline. Swabs were agitated vigorously, and excess fluid was expressed. DNA from PBLs and from the cells in nasal secretions was extracted by protease K digestion and phenol-chloroform extraction and precipitated by ethanol (12, 28). Purified DNA was quantitated by spectrophotometry at 260 nm.

PCR.

Viral DNA in PBLs and in nasal secretions was monitored by PCR as described previously (10). In order to control any contamination by PCR products, DNA sample preparation and PCR reagent assembly were conducted in a room separated from the room used for PCR amplification and product analysis. Positive OHV-2 DNA and no-DNA controls were included in each set of PCR amplifications to control for amplification inhibition or reagent contamination.

CI-ELISA.

OHV-2 antibody was assayed by the previously described CI-ELISA, which employed a monoclonal antibody specific for the 15A epitope of MCF virus (9). An animal was considered seropositive for anti-MCF virus antibody if its serum inhibited binding of the monoclonal antibody more than 3 standard deviations beyond the mean of a panel of previously defined negative sheep sera.

RESULTS

OHV-2 antibody and DNA in fetuses and in presuckling lambs.

No OHV-2 antibody was detected in any of four fetuses obtained in late gestation from OHV-2-positive ewes. Moreover, no viral DNA was detected in their PBLs or in lymph nodes by PCR (data not shown).

Of a total of 118 serum samples from presuckling lambs born to OHV-2-positive ewes, none was detected positive for the virus by CI-ELISA. The PBLs of 77 of these lambs were examined by PCR; only four samples were positive for viral DNA. Two of the signals were suspected to be due to sample contamination in the laboratory, since the lambs were negative for viral DNA on subsequent bleeding. The remaining two lambs were twins; they were consistently positive by PCR.

Development of anti-OHV-2 antibody in lambs.

Ninety-three percent of 70 lambs born to OHV-2-positive ewes acquired maternal anti-MCF viral antibody from colostrum as expected. The maternal antibody remained detectable until about 2.5 months of age (Fig. 1 and 2). Active antibody synthesis resulted in detectable levels in lambs, beginning at 6 months of age. A total of 50% of the lambs seroconverted at about 7 to 8 months of age, and 80 to 90% of lambs seroconverted by 1 year of age (Fig. 1 and 2). This is consistent with the percentage of seropositivity in adult sheep (9).

FIG. 1.

OHV-2 DNA in PBLs (open squares) and in nasal secretions (open triangles) and serum antibody (open circles) in lambs from OHV-2-infected ewes under normal husbandry conditions over a period of a year (n = 43 for first 3 months; n = 26 thereafter).

FIG. 2.

OHV-2 antibody in lambs separated from the flock at weaning (open squares) (n = 12) and under normal husbandry conditions (open circles) (n = 15).

Appearance of OHV-2 DNA in PBLs and nasal secretions of lambs.

After nursing, the PBLs from about 10 to 20% of lambs contained low levels of viral DNA detectable by PCR and 5 to 8% of lambs contained detectable viral DNA in nasal secretions. During the first 3 months of life, PCR signals were relatively weak and sporadic, often occurring in different lambs on subsequent bleedings. The number of PCR-positive lambs rose sharply at 3.5 months of age to 87%, and the PBLs of all lambs (100%) yielded strong PCR signals at 5.5 months of age (Fig. 1). In contrast, the percentage of lambs in which the nasal secretions were PCR positive was 5% at 3.5 months and 33% at 5.5 months of age. The peak prevalence of viral DNA in nasal secretions (88%) was reached at 7.5 months of age, declining rapidly thereafter (Fig. 1).

Isolation of lambs at different time points.

In experiment 1, a group of 12 lambs was weaned at 3 months of age and raised in isolation from all other sheep. At 13 months of age, all animals (12 of 12) remained free of detectable levels of both OHV-2 DNA (data not shown) and antibody (Fig. 2). In contrast, 93% (14 of 15) of lambs in the control group, which were returned to the flock following the weaning period, had seroconverted by 13 months of age (Fig. 2). In experiment 2, which was designed to roughly define the age at which infection occurred, five of five lambs weaned at 2.5 months of age and isolated from the flock remained negative for both viral DNA and antibody. The other half of the group (n = 5), returned to the flock after the weaning period, were PCR positive at 3.5 months of age. No difference was observed between the two groups that were weaned at 3.5 months of age, one of which was separated from the flock and the other of which was returned to the flock. All animals in both groups were PCR positive at 3.5 months of age (Fig. 3).

FIG. 3.

Agarose gel electrophoresis of PCR products amplified from PBL DNA of lambs at 2.5 (A) and 3.5 (B) months of age. Lanes 1, 100-bp DNA ladder; lanes 2 to 6, samples from lambs isolated from infected sheep at 2.5 (A) or 3.5 (B) months of age; lanes 7 to 11, samples from lambs weaned at 2.5 (A) or 3.5 (B) months of age and returned to the sheep flock immediately after the weaning period. Lambs whose samples are in lanes 2 to 6 are twins of the lambs whose samples are in lanes 7 to 11. Lanes 12, no-DNA control; lanes 13, OHV-2-positive DNA control.

DISCUSSION

The transmission pattern of OHV-2 in sheep differs significantly from that of AHV-1 in wildebeests. Although some transplacental infection occurs in wildebeests (16, 17), most calves are infected shortly after birth (16, 17). Infection ensues, which results in active viral shedding to the environment in naso-ocular secretions, primarily during the first 3 months of life, the young calves apparently serving as the predominant source of infection. In this study, 118 of 118 serum samples from presuckling lambs were free of detectable anti-MCF viral antibody and 97% of 77 presuckling lambs were free of detectable viral DNA in PBLs. The data strongly suggest that transplacental transmission of OHV-2 is relatively uncommon in sheep. The fact that weaning and separation of lambs from infected sheep at 2.5 months of age prevented the infection implies that newborn lambs are not a source of virus for transmission to clinically susceptible species or to other sheep.

Previous studies showed that colostrum and milk samples from infected ewes were strongly positive for OHV-2 DNA (10), which suggests that mammary secretions could serve as an important source of infection for newborn lambs. For the first 2 to 3 months after nursing, viral DNA sequences were sporadically detectable in the PBLs of about 10 to 20% of the lambs on any given occasion. Most of these positive signals, however, were weak and inconsistent. The data from the separation experiments showed that the lambs isolated from the OHV-2-positive flock at 2.5 or 3 months of age remained negative, even though some exhibited viral DNA on occasion during the first few weeks of postnatal life. This suggests that PCR signals detected for some lambs during the first several weeks originated from passively transferred maternal leukocytes (24, 29), which were eventually eliminated, leaving the lambs uninfected.

The fact that lambs are not infected during the neonatal period could be related either to the presence of passively acquired maternal immune components or to age dependency of susceptibility at a cellular level. Although nonimmune factors in milk may be involved in inhibition of viral binding or entry (7, 11, 15), these are unlikely mechanisms in OHV-2 infection, since no differences in the course of infection were observed between weaned and unweaned groups of lambs (unpublished data). Studies are under way to determine whether maternal immunity or age-specific factors play significant roles in determining susceptibility.

The present study showed that the majority of lambs seroconverted between 7 and 9 months of age, which is consistent with the previous observations of a low rate of seropositivity among immature sheep (8, 9). The seroconversion closely paralleled the rise of viral DNA in nasal secretions but not its appearance in peripheral blood lymphocytes. This suggests that viral replication in lymphocytes of these lambs may be highly restricted, not expressing sufficient viral antigens to stimulate a humoral immune response. The results obtained from a transmission study employing inoculation of viable leukocytes from OHV-2-infected sheep into OHV-2-negative sheep also support the concept that OHV-2 replication in lymphocytes is highly restricted (unpublished data). The temporal correlation between the appearance of viral DNA in nasal secretions and seroconversion strongly suggests that nasopharyngeal epithelial cells support productive infection by OHV-2. Explanations for the fact that the appearance of viral DNA in nasal secretions and seroconversion were delayed several months after the initial detection of viral DNA in lymphocytes could include age-related susceptibility or permissiveness of certain cell populations such as nasopharyngeal epithelial cells. Admittedly, no proof exists that the PCR signals from the nasal secretions actually represent infectious virus and not latently infected cells. More direct methods, such as electron microscopy for viral particles and immunochemistry for viral antigens, should be applied for clarification. However, the intense signals observed in nasal secretions and their temporal association with antibody synthesis are not likely to be due to the presence of latently infected lymphocytes in the secretions. Recent observations in this laboratory with a quantitative PCR assay indicate that the number of copies of OHV-2 per microgram of DNA in cells from nasal secretions exceeds that of PBLs by several hundredfold (unpublished data).

A recent study by Baxter et al. (2) reported that lambs were infected at very early ages, which is in conflict with the data in this study. The reason for this discrepancy is not clear. Since PCR signals are routinely detected in a certain proportion of lambs due to passively transferred maternal lymphocytes from infected ewes (10), it is possible that the PCR signals observed in that study reflected these passively transferred maternal leukocytes, instead of an active infection. The high copy number of OHV-2 DNA (5 × 10⁴ copies/μg of DNA) in the cells from the colostrum of infected ewes (unpublished data) supports this possibility.

The traditional recommendation for controlling SA-MCF has been to keep sheep away from cattle, deer, or other clinically susceptible species. Separation, however, while practical for some situations, is not for others, particularly for game farms, petting zoos, and certain sheep-cattle range operations. Results of this study indicate that OHV-2-free sheep can be established by separation of lambs at the proper time (approximately 2.5 months of age), which offers an important alternative approach to the control of SA-MCF. Studies to better define the critical time point for the separation and to develop monitoring and security measures are ongoing. Control of SA-MCF by the production and use of OHV-2-free sheep should, at the very least, prove effective for zoos, small game farms, and other small operations in which accidental contact with infected animals can be effectively prevented.