A female cria (cria 1) was born into an alpaca herd located in Pennsylvania. It was born 3 weeks premature and appeared clinically normal except for a less than typical birth weight. Routine evaluation of a blood sample obtained 2 days after birth revealed that the PCV was 29% (lower reference limit, 27%), hemoglobin concentration was 11.5 g/dL (lower reference limit, 11.9 g/dL), and WBC count was 14.9 × 103 cells/mL (reference range, 8 to 21 × 103 cells/mL). Serum total protein concentration was 6.5 g/dL (reference range, 5.7 to 7.3 g/dL), and IgG concentration was 2,000 mg/dL (800 mg/dL [lower reference limit] was considered adequate to prevent neonatal diseases associated with failure of passive transfer). Platelet count was judged as adequate. When the cria was approximately 10 weeks old, it developed a moderate mucopurulent nasal discharge and had a high rectal temperature (40.1° C [104.2° F]). Analysis of a blood sample obtained at that time revealed that the PCV was 17.4%, hemoglobin concentration was 6.4 g/dL, and WBC count was 10.4 × 103 cells/mL. Serum total protein concentration was 6.0 g/dL. The cria was administered penicillin G procaine (6,600 U/kg [3,000 U/lb], IM, q 24 h) for 5 days, flunixin meglumine (0.55 mg/kg [0.25 mg/lb], SC, q 24 h) for 5 days, and florfenicol (20 mg/kg [9.1 mg/lb], IM, q 48 h) for 3 treatments. The cria initially responded to treatment, but during a 5- to 10-day period, it slowly began to relapse and became febrile and developed mucopurulent nasal discharge. Five weeks after the first treatment, the owner requested reevaluation of the cria's clinical condition because the nasal discharge was more pronounced and a bilateral mucopurulent ocular discharge had also developed.
Between 15 and approximately 21 weeks of age, the cria was examined on several occasions in an attempt to provide a diagnosis for the clinical condition. The cria progressively developed more severe signs of an infection of the upper portion of the respiratory tract (ocular and nasal discharges), low-grade pyrexia, lethargy, and inappetence. Further, the cria failed to gain weight as anticipated. During this period, the cria was treated with various antimicrobial preparations either alone or in combination. These treatments included sulfadimethoxine (55 mg/kg [25 mg/lb], PO, once initially followed by 28 mg/kg [12.7 mg/lb], PO, q 24 h for 5 days), ceftiofur sodium (2.2 mg/kg [1.0 mg/lb], SC, q 24 h for 5 days), gentamycin sulfate (4.4 mg/kg [2 mg/lb], IM, q 24 h for 5 days), and oxytetracycline hydrochloride (11 mg/kg [5 mg/lb], PO, q 12 h for 5 days). Vitamin supplements including thiamine (8.8 mg/kg [4 mg/lb], SC, q 24 h for 5 days) and B complex vitamins (unknown dose, PO, q 24 h on a continuous basis) were administered. On 3 occasions, the cria received lactated Ringer's solution (12 mL/kg [5.5 mL/lb], SC and IV). Following each antimicrobial treatment, there was temporary improvement of the cria's condition. After treatments were discontinued, the cria slowly regressed to its previous clinical status and no permanent improvement was evident. During this treatment period (ie, between 15 and approx 21 weeks of age), blood samples were collected on 10 occasions and CBCs were performed; overall, mean ± SD PCV was 14.7 ± 2.5% and mean ± SD WBC count was 3.96 × 103 ± 2.5 × 103 cells/mL. Hemoglobin concentration was assessed twice (values of 5.9 and 6.0 g/dL). On 4 occasions, platelet counts were performed and considered to be adequate. Serum IgG concentration was evaluated again and was 1,800 mg/dL.
Immediately after the last examination of the cria, blood samples from the cria, its dam, and 15 other alpacas in the herd were collected and submitted to the diagnostic laboratory for virus isolation. Bovine viral diarrhea virus was isolated from BCCs from the cria as well as from a 4-year-old male alpaca. Virus was not recovered from samples collected from the remaining animals. Thirty-six days later, blood samples from the cria and the 4-year-old alpaca were reevaluated via virus isolation. Virus was isolated only from BCCs obtained from the cria; by use of rt-PCR assay, the virus was identified as BVDV genotype 1. On the basis of these findings, persistent infection with BVDV was diagnosed in the cria, whereas the adult alpaca was considered to have been acutely infected with the virus and was now in a recovery stage.
Over the following weeks, the cria continued to have signs of disease of the upper portion of the respiratory tract, inappetence, lethargy, low-grade pyrexia, and a progressive unthrifty condition. During the entire course of observation, the cria did not have clinical signs of a disease of the lower portion of the respiratory tract or the alimentary tract. The owner treated the cria periodically with ceftiofur sodium (2.2 mg/kg, IM, q 24 h) for 5 to 6 days when its condition became more severe. At approximately 24 weeks of age, all treatment was withdrawn. The clinical condition of the cria became increasingly grave, and 15 weeks later, the owners requested euthanasia and necropsy of the cria.
During necropsy, gross pathologic examination revealed the presence of congested blood vessels over the entire serosal surface of the intestinal tract with more intense hyperemia involving the jejunal serosa. There were petechial hemorrhages over the serosal surfaces of the kidneys and an excessive amount of peritoneal fluid. Blood samples for serologic evaluation and virus isolation were submitted to the diagnostic laboratory. In addition, samples of lungs, spleen, jejunum, jejunal mesenteric lymph node, kidneys, abdominal cavity fluid, and bone marrow were submitted for virus isolation. Bovine viral diarrhea virus was isolated from all samples. Virus neutralization tests performed on samples of serum from the cria did not detect antibodies against BVDV genotypes 1 or 2 at the 1:4 dilution (lowest dilution evaluated).
Twenty-two days after cria 1 was euthanized, another female cria (cria 2) was born into the herd. Analysis of a blood sample obtained 24 hours after birth revealed that the PCV was 25.5%, hemoglobin concentration was 9.9 g/dL, and WBC count was 6.4 × 103 cells/mL; total serum protein concentration was 5.1 g/dL. When cria 1 was born, the dam of cria 2 was at approximately 49 days of gestation and had continual contact with cria 1 throughout the remainder of its pregnancy. Although cria 2 had no overt clinical signs of disease, the owners were aware that it was failing to gain weight as anticipated. When cria 2 was 6 weeks old, a blood sample was submitted to the diagnostic laboratory; virus isolation and rt-PCR procedures revealed that the BCCs were positive for BVDV. When the owners were informed that BVDV had been detected in BCCs from cria 2 and that the animal could be persistently infected, they requested immediate euthanasia. Cria 2 underwent necropsy 7 days after the first sample was obtained, and additional blood samples for virus isolation and serologic evaluation were submitted at that time. Via virus isolation, BVDV genotype 1 was identified in the BCCs. In addition, immunohistochemical staining revealed that biopsy specimens of skin of the ear and perineal area contained antigens of BVDV in cells of the germinal layers of the epidermis and hair follicles. Serum did not contain detectable VN antibodies against either BVDV genotypes 1 or 2 at the 1:4 dilution. Although persistent BVDV infection in cria 2 was suspected, a diagnosis could not be verified because the interval between collections of the BVDV-positive blood samples was of insufficient duration. Assuming that cria 2 was persistently infected, it is noteworthy that both crias 1 and 2 were infected with BVDV genotype 1.
On the same day as the necropsy of cria 2 was performed, blood samples were collected from all remaining members of the herd for virus isolation and serologic evaluation. At this time, there were 15 camelids in the herd because 1 alpaca had been relocated by its owner. Virus was not detected in any of the BCC samples. One alpaca did not have serum antibodies against BVDV, but the other 14 alpacas had antibodies against BVDV genotype 1 (log-transformed mean serum VN antibody titer, 1:280; range, 1:16 to 1:2,048) and BVDV genotype 2 (log-transformed mean serum VN antibody titer, 1:94; range, 1:8 to 1:512).
Discussion
Criteria for diagnosis of persistent infection with BVDV in alpacas and other New World camelids have not been established; it is proposed that the model used for cattle be followed. Confirmation of a diagnosis of persistent BVDV infection is based on the fact that when a normal adult bovid becomes infected with BVDV, the duration of viremia is typically 4 to 8 days.1,2 On this assumption, one means by which a persistently infected animal can be identified is detection of virus in serum or BCCs on 2 occasions with a minimum interval of 21 days between the sample collections.3–5 Another procedure used to identify persistently infected bovids is immunohistochemical staining of skin biopsy specimens to detect BVDV antigen.6–8 Because germinal cells of the epidermis and hair follicles may retain BVDV antigens and yield positive immunohistochemical test results in a low percentage of nonpersistently infected cattle for a variable period of time following acute infection,9 some diagnosticians recommend that a confirmatory test be performed 30 days following an immunohistochemical staining procedure. Confirmation of findings may be accomplished via detection of BVDV in BCCs, detection of viral genome in BCCs by use of rt-PCR assays, or other methods of virus identification.9,a
Persistently infected cattle do not develop neutralizing antibodies against the specific antigenic strain of BVDV with which they are infected but will develop antibodies against heterologous antigenic strains.2,3 If serum antibodies are detected in persistently infected cattle, titers are typically low; the antibodies are believed to be a result of either previous natural infection or vaccination with a heterologous antigenic strain of virus.10 Because the presence of VN antibodies is an inconsistent finding, it cannot be used to diagnose persistent infection in individual bovids; however, a high percentage of cattle that have serum antibodies against BVDV in an unvaccinated herd may indicate the presence of a persistently infected animal.2
The pathogenesis of persistent infection in a bovine fetus has been well characterized.3,11,12 Several conditions must be present, the first of which is that the dam develops viremia with a noncytopathogenic strain of BVDV during gestation. Trophoblastic cells must be in intimate contact with maternal cotyledons (ie, the embryo must have undergone implantation). Trophoblastic cells must become infected (ie, have receptors for the virus). The strain of virus should not be so highly virulent that it induces fetal death. Lastly, infection must occur during the self-recognition phase of immune system ontogeny. This phase terminates at approximately 120 days of gestation in bovids.1,3,13 It is proposed that a similar pathogenesis mechanism must prevail for persistent infection to develop in fetuses of New World camelids. Because the gestation period of alpacas and llamas is longer than that of bovids (approx 345 days vs 284 days), allowances must be made for a longer self-recognition phase. If the ontogenesis of the immune system for New World camelids is proportionally similar to that of bovids, it is estimated that fetal New World camelids will be susceptible to development of persistent infection until approximately 145 days of gestation.
On the basis of published information14 and experiences of the senior author and other diagnosticians, it has been thought that viremia seldom develops in New World camelids following infection with BVDV; if viremia did develop, it was believed to be of short duration.b Likewise, following infection of a New World camelid, BVDV does not appear to be transmitted easily to other members in the herd.14,c Results of seroprevalence studies15–18,d have supported this observation in that a low percentage of New World camelids have antibodies against BVDV. These past concepts of BVDV infection in New World camelids suggested the virus was not well adapted to this species. Findings of other studies14,d have also indicated that New World camelids have a higher risk of becoming infected with BVDV when they are in contact with cattle. These concepts may have a bearing on the results of a study by Wentz et al15 in which persistent infection was not induced in fetuses following experimental exposure of pregnant llamas to an aerosol preparation of BVDV. Even though the dams developed viremia, the authors concluded that the virus did not cross the placental barrier.15 This observation may be attributable to the fact that trophoblastic cells did not possess proper receptors for the strains of viruses used in the study. On evaluation of the aforementioned and the fact that there have been relatively few reports14,19,20 of disease attributed to BVDV infection in New World camelids, many veterinarians have concluded that BVDV infection was of minor importance in this group of animals. These past concepts may have to be reevaluated following the report of a persistently infected alpaca in Canada21 and the apparent diagnosis of persistent infection in alpacas in various regions of the United States.22
The mutagenic properties of the BVDV genome have been well documented.23–25 As virus replicates, genomic variants (quasispecies) may be produced.26–28 Within the BVDV genome, there are several genes that encode for structural glycoproteins of the outer capsid.29,30 Changes within these genes may be expressed as a variation in the antigenic profile of the virus. Also, alterations at this location may allow virus to attach onto specific receptors on the surface of different cells within the host or more efficiently attach to cells of a different species for which the parent strain was not well adapted.26 Such diversity may explain why some strains of BVDV readily infect trophoblastic cells and facilitate development of persistent infection in fetuses, as illustrated by the findings in the crias of this report. In contrast, other strains may not possess this characteristic and fail to infect trophoblastic cells (eg, the virus strains used by Wentz et al15 in their attempt to experimentally induce persistent BVDV infection in fetal llamas). It should be noted that in their study, Wentz et al inoculated 4 llamas (at 67, 68, and 102 days of gestation) with 3 strains of BVDV, 1 of which was isolated from an aborted llama fetus. Each pregnant llama was believed to be infected at the proper stage of gestation (ie, after implantation of the fetus, which would allow trophoblastic cells to become infected). Because of an apparent increase in the diagnosis of persistent infection in alpacas,22 it is speculated that certain strains of BVDV (quasispecies) may now be adapting to New World camelids (ie, readily infecting these animals, which results in viremia and infection of trophoblastic cells); research into the molecular composition of these isolates may assist in evaluating this speculation.
Calves that are persistently infected with BVDV often begin to show the effects of BVDV infection after colostral antibodies are dissipated.1,31,32 Although some persistently infected calves appear clinically normal, others develop a chronic respiratory or alimentary tract disease, pyrexia, inappetence, lethargy, and failure to gain weight (compared with their contemporaries).1,32 It should be noted that cria 1 did not develop clinical signs of disease until after 10 weeks of age (presumably after colostral antibodies were dissipated). The major clinical features consisted of respiratory tract disease with concurrent anemia and leukopenia, and a progressive unthrifty condition.
An additional observation concerning the pathogenesis of BVDV infection in cattle may have application in understanding the persistent infection of New World camelids. When a persistently infected calf is in contact with other members of the herd, most herd mates become infected with the specific antigenic strain of virus that is being shed by the persistently infected individual. As a result, most contact animals develop high serum concentrations of VN antibodies against the virus. These animals appear to develop some immunity against the strain of virus in question because they do not develop clinical signs of disease after subsequent exposure to the persistently infected calf. However, although pregnant cows have such serum antibodies (as indicated by results of VN tests), they still can become infected following exposure to the persistently infected individual. Infection may lead to viremia or presence of virus in WBCs. This, in turn, allows virus access to trophoblastic cells and infection of the fetus ensues. If the fetus is at the susceptible stage of gestation, it may become persistently infected.1,3,33 Because this situation perpetuates the presence of persistently infected bovids in the herd, the only method whereby persistent infection can be eliminated from cattle herds is to test all animals and remove those that are persistently infected.1,3,33 During the 8 to 9 months after persistently infected animals have been removed, all newborn calves should be assessed for persistent infection.33 Compared with cattle, the duration of gestation in alpacas is longer and it is recommended that newborn crias are evaluated for persistent infection for at least 11 months following the removal of a persistently infected camelid from the herd.
ABBREVIATIONS
| BVDV | Bovine viral diarrhea virus |
| BCC | Buffy coat cell |
| rt-PCR | Reverse transcriptase-PCR |
| VN | Virus neutralizing |
Brodersen B, Nebraska Veterinary Diagnostic Laboratory, University of Nebraska, Lincoln, Neb: Personal communication, 2005.
Evermann J, Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, Wash: Personal communication, 1993.
Meyer C, Veterinary Practitioner, Grants Pass, Ore: Personal communication, 1994.
Picton R. Serologic survey of llamas in Oregon for antibodies to viral diseases of livestock. MS thesis, College of Veterinary Medicine, Oregon State University, Corvallis, Ore, 1993.
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