Bovine viral diarrhea virus in alpaca: abortion and persistent infection

Transcription

1 589 J Vet Diagn Invest 17: (2005) Bovine viral diarrhea virus in alpaca: abortion and persistent infection Susy Carman, 1 Nancy Carr, Josepha DeLay, Mohit Baxi, Dirk Deregt, Murray Hazlett Abstract. An alpaca herd in eastern Ontario experienced vague signs of illness, including anorexia and lethargy in 9 animals, 2.5 months after the addition of a chronically ill cria and his dam to the farm. Subsequently 2 alpaca had early pregnancy loss; one aborted at 5.5 months gestation and the other at 7 months gestation. Seventeen were found to have serum antibody to bovine viral diarrhea virus (BVDV), with highest titers to BVDV type 1. The fetus that was aborted at 5.5 months gestation, 3 months after the clinical outbreak, was found to be positive for BVDV on immunohistochemical staining, and noncytopathic BVDV type 1b was isolated. Of the 13 cria born alive that season, a single male underweight alpaca cria, born 9 months after the clinical illnesses, was infected with BVDV type 1b. The cria was positive for BVDV at birth, at 3 and 26 days of age and continued to be positive for noncytopathic BVDV using virus isolation, nested reverse transcription PCR, antigen detection ELISA, and immunohistochemical staining until euthanasia at 46 days of age. The cria remained serum antibody negative to both BVDV type 1 and type 2. A diagnosis of persistent infection was made. This is the first report describing persistent infection with BVDV in an alpaca cria. Key words: Alpacas; bovine viral diarrhea virus; new world camelids; persistent infection. Cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) are the primary source of virus spread within and between cattle herds. Reproductive losses are the most economically important consequence. 6 Depending on the stage of gestation when infection of the susceptible dam and fetus occurs, one may observe failure of fertilization; early pregnancy loss; abortion; persistent infection with immunotolerance; congenital anomalies in the central nervous system with ocular, cutaneous, and skeletal defects; and stillbirth, or birth of weak neonates. 6 BVDV infection has been identified in many nonbovine species, including sheep, goats, swine, exotic ruminants, deer, old world camelids, and among new world camelids (NWCs) in llamas and alpaca. 1,5 NWCs are rarely seropositive to BVDV, unless exposed to cattle, 1,8,11 and it has been suggested that transmission of BVDV in NWC is inefficient. 8 Although experimental infection of 4 seronegative llamas with BVDV at 67 to 102 day gestation caused no signs of illness in dams and no evidence of fetal infection, 12 abortions owing to BVDV have been reported in a llama 1 and in alpaca. 5 Both noncytopathic (ncp) and cytopathic BVDV have been isolated in combination from a newborn llama cria delivered by caesarian section and from a 15-month-old male llama with lymphoid depletion and thymic atrophy, 1 suggesting persistent infection. Little is known about BVDV infection and its consequences in NWCs. The natural infection of an alpaca herd with BVDV resulting in clinical illness in adults, early pregnancy loss, abortion, and the birth of a PI alpaca cria on an eastern Ontario farm is described. From the Animal Health Laboratory, Laboratory Services Division, University of Guelph, Guelph (Carman, DeLay, Hazlett), Ontario, Canada, Elginburg (Carr), Ontario, Canada, and Canadian Food Inspection Agency, Animal Diseases Research Institute, Lethbridge, AB, Canada (Baxi, Deregt). 1 Corresponding Author: Dr. Susy Carman, Animal Health Laboratory, Laboratory Services Division, University of Guelph, Box 3612, Guelph, ON, N1H 6R8, Canada. Alpacas were housed (Table 1) in 2 separate groups in one barn (west and south enclosures), adjacent to but separated by a partition from a third group of dams with cria (east enclosure). Males were housed in a fourth location. There were a total of 52 alpaca on the farm. The herd had no known contact with either cattle or sheep. This case study was initiated when a 21-month-old female alpaca (alpaca 1) aborted a 5.5-month fetus. A second alpaca (alpaca 11) aborted later at 7 months gestation, but this fetus was not tested. The only recent animal introductions to this herd were a dam (alpaca 12) and a chronically ill cria (cria 1), which had been introduced into the group of dams with cria housed in the east enclosure (Table 1) 2.5 months before a vague herd illness (with signs of anorexia and lethargy in 9 adult alpaca) and 5.5 months before the time of alpaca 1 s abortion. This cria was in poor body condition on arrival at this Ontario farm at 3 months of age, with intermittent diarrhea, pneumonia, and nasal discharge, and died at 8 months of age despite repeated antibiotic therapy. This cria was not necropsied. The cria s dam had been on 4 different farms during her pregnancy, including an Alberta farm that experienced numerous abortions and another Alberta farm that had two stillborn cria and a cria that died at 2 days of age. Alpaca on these farms were found to have antibody to BVDV, but none had known contact with cattle or sheep (personal communication with farm owners). No significant gross, histological, or bacteriological abnormalities were identified in alpaca 1 s aborted fetus. Immunohistochemistry for BVDV (IHC-BVDV) was performed on formalin-fixed, paraffin-embedded tissue sections 7 of lung, kidney, heart, liver, and placenta that were pretreated with Proteinase K a for 10 minutes at room temperature. Automated IHC procedures used anti-bvdv monoclonal antibody 15C5 cell culture supernatant b at a 1:5 dilution with 60-minute incubation at room temperature as primary antibody and goat anti-mouse immunoglobulin conjugated to a horseradish peroxidase labelled polymer c as the secondary antibody, with 30-minute incubation at room temperature.

2 590 Table 1. Serum virus-neutralizing antibody titers to bovine viral diarrhea virus (BVDV) in alpaca following introduction of the sickly Cria 1. Alpaca No./location* Titer to BVDV type 1a (NADL strain) Females from west enclosure 1a,b,c 2b,d 3b,c,e 4b,c,e 5b,c,f 6b,f 7b 8b 1:512 1:768 1:768 1:256 Young males in the south enclosure 9b 10 b, c Females from east enclosure 11 a 12 b, f, h 13 g 14 b 15 f 16 f 17 i 18 i 19 f 1:3072 1:1024 1:2048 1:32 Titer to BVDV type 2 (NVSL-125c strain) 1:48 1:96 1:8 1:394 1/2 1:16 1:48 1:96 1:8 Adult male in the male barn 20 b Stage of gestation at time of presumed exposure 70 days 65 days 80 days 75 days 170 days 180 days 30 days *a dams of aborted fetuses; b animals negative on BVDV antigen ELISA; c animals with clinical illness; d dam of the persistently infected Cria 2; e animals with early pregnancy losses; f dams with live uninfected cria born in the same year as Cria 2; g animal that left the farm before the arrival of the sickly Cria 1; h dam of sickly Cria 1 imported into the head at 3 months of age; i juveniles 1 year old. Nova red d was used as chromogen. BVDV was identified in cardiocytes, epithelium of kidney and lung, lamina propria and tunica muscularis of small intestine, and vascular smooth muscle of heart, kidney, and lung. Tissues from alpaca 1 s aborted fetus were also tested for BVDV by using virus isolation. 2 The ncp BVDV was isolated in secondary bovine spleen cells from skin removed from between the ears, but not from lung, liver, kidney, or heart. The virus isolate (designated 24913) was determined to be BVDV type 1 by using nested reverse transcription (nrt)-pcr. 4 The E2 gene of the BVDV was reverse transcribed, amplified by PCR, and cycle sequenced by methods previously described 3 and compared to those in GenBank. The virus was identified as BVDV type 1b, clustering with Osloss, Hastings, and CP7 (Fig. 1). After the identification of BVDV in alpaca 1 s aborted fetus, the Ontario farm submitted sera from 17 adult alpaca and 2 juvenile alpaca from the 4 different housing areas, and from a single female (alpaca 13) that had left the farm before the arrival of the sickly cria (cria 1), for determination of BVDV serum antibody titers (Table 1). These alpaca had assorted histories, including no illness, clinical illness, early pregnancy loss, abortion, or delivery of live cria. Seventeen of the 20 animals had antibody to both BVDV type 1a (NADL strain) and BVDV type 2 (NVSL 125c strain), using serum in standard microtiter virus-neutralization assays. 2 The animals with antibody to BVDV showed higher titers to BVDV type 1, which is consistent with the recovery of BVDV type 1b from alpaca 1 s aborted fetus. The alpaca with the highest antibody titers to BVDV type 1 (1:3072) was alpaca 11. She was subsequently moved to a different farm at 30 days gestation and was the dam of the fetus aborted later at 7 months gestation that was not tested for BVDV. The serum from the alpaca (alpaca 13) that had left the farm before the arrival of the sickly cria (cria 1), and before the clinical illnesses in adults, was negative for antibody to BVDV. Sera from 13 of these alpaca (Table 1) were negative for BVDV in a BVDV antigen detection ELISA. e Thirteen cria born subsequent to the initial abortion were tested for BVDV at birth by using EDTA blood, serum, or plasma in a nrt-pcr. 4 Only one cria born after the abortions was infected with BVDV. This single BVDV infected male cria (cria 2) was born from a dam (alpaca 2) that had not been clinically ill (Table 1). He was from the same cohort of cria as was alpaca 1 s aborted BVDV infected fetus. He had a low birth weight (12 pounds) for the Ontario farm (average, 18.1 pounds), chronic diarrhea, episodic lacrimation, and a prominent umbilical hernia. His fleece was slightly less dense than that of other cria of the same age, with longer hair-like fibers from primary follicles extending past the woolly fibers from secondary follicles. Hairy fleece has been reported in PI lambs with border disease virus. 9 Over the course of 6 weeks, multiple samples from cria 2 were tested for BVDV. The ncp BVDV was isolated 2 from the placenta and at 3 and 26 days of age from buffy coat cells. BVDV type 1 was detected by nrt-pcr 4 in both buffy coat cells and plasma. Antibodies for BVDV type 1 and type 2 were not detected using serum in virus-neutralization assays. 2 A diagnosis of PI with BVDV was made, cria 2 was euthanized at 46 days of age, and multiple samples were tested for BVDV. At this time he was positive for BVDV antigen detection ELISA using serum and a fresh ear notch. The ncp BVDV was isolated 2 from buffy coat cells, plasma, serum, cerebrum, cerebellum, thymus, mesenteric lymph node, thyroid, kidney, and spleen, but not from lung or liver. BVDV type 1 was detected by using nrt-pcr 4 in both plasma and serum. All ncp BVDV isolates were typed as BVDV type 1 by using nrt-pcr. 4 The ncp BVDV type 1 isolate recovered at 46 days from cria 2 was also sequenced 3 and compared with the BVDV isolated from alpaca 1 s aborted fetus (Fig. 1). The isolate from cria 2 was also determined to be BVDV type 1b. The 2 viruses differed by only 4 nucleotides over the entire E2 gene, resulting in 2 amino acid changes at positions 61 (Glu to Gly) and 250 (Val to Glu), with numbering relative to the Singer strain of BVDV. 3 This indicated that they represented variants of the same virus. In addition, this was a unique virus not matching any other in GenBank. Both BVDV type 1a and 1b strains have been

3 591 Figure 1. Phylogenetic tree based on the E2 gene of bovine viral diarrhea virus (BVDV) showing the relationship of the BVDV isolated from alpaca 1 s aborted fetus to other pestiviruses. isolated from NWCs (llamas). 12 Cria 2 continued to be antibody negative for both BVDV type 1 and BVDV type 2 at 46 days. Demonstration of BVDV in this cria over a 6-week period, without development of antibody to BVDV, confirms persistent infection. There were no significant findings on gross postmortem examination of the PI cria (cria 2), except for a 1-cm umbilical hernia and unformed colonic content. No parasites were found on fecal flotation. Growth plates of long bones appeared normal. Microscopically, there was occasional necrosis of ganglion cells in the retina of the eye. In some brain sections, there were foci containing scattered remnants of cellular debris, suggesting lysis of neurons or microglia. IHC staining for BVDV antigen was seen in many tissues, including epithelium of oral cavity, stomach, kidney, thyroid gland (Fig. 2a), and skin (Fig. 2b), including pinna, with staining patterns consistent with that described for PI cattle. 10 There was also staining in placental trophoblasts, retina, histocytes of lymph node and thymus, and thymic epithelium. Sparse staining was seen in brain and spinal cord. On this Ontario farm, there were 9 alpaca with clinical signs of illness, 2 alpaca with early pregnancy losses at 75 to 80 days of gestation and a second abortion at 7 months gestation that were not tested for BVDV. The abortion associated with BVDV 3 months after the clinical illness in the herd, and the subsequent birth of a BVDV PI cria 5

4 592 Figure 2. Thyroid gland, A, and skin, B, of cria 2 stained for bovine viral diarrhea virus (BVDV) by immunohistochemistry using Nova red chromagen. The brown-red color demonstrates location of BVDV antigen. months later, suggests the clinical disease in this herd of alpaca might all have been attributable to BVDV. The stage of gestation at the time of presumed exposure of 7 alpaca to BVDV is listed in Table 1. The mean gestation period for alpaca is 342 days (range, 335 to 356 days). The PI cria was born from a first parity dam at 338 days of gestation. Alpaca 2, the dam of this cria, likely became infected at the time of the clinical illness in her cohorts, at about 65 days gestation. Alpaca 1, the dam of the 5.5-month aborted fetus, also likely became infected at about 70 days

5 593 gestation. This stage of gestation is similar to the timing of experimental infections of seronegative llamas at 67 to 102 days gestation, in which no signs of illness were observed in dams and no evidence of fetal infection was identified. 12 Twelve cria on this Ontario farm were born uninfected, the dams of 6 had high levels of serum antibody to BVDV after the first abortion and before their cria s birth. The BVDV antibody status of the dams of the other 6 cria was not determined. The history suggests that BVDV infection was likely introduced to the farm by the sickly cria, cria 1, who may have been PI with BVDV. Because the virus isolated in this study does not represent a unique pestivirus species, it is likely to have originated from a bovine as noted by others 12 and infected an alpaca herd. Conversely, PI alpaca families and maintenance of PI in some alpaca herds cannot be ruled out at this time. It is uncertain, but the efficiency of production of PI in alpacas may be lower than in cattle. 8 However, it is possible that these viruses will evolve over time to become more efficient in producing both clinical disease and PI. Cria with PI BVDV represent a hazard to the alpaca industry, in which dams with cria at foot are commonly sent to different farms for breeding. These cria have great potential to infect naïve alpaca herds and cause economic losses. It has been suggested that BVDV infection be considered in llamas with abortion, diarrhea, unthriftiness, and weight loss. 1 This recommendation should now be extended to alpaca based on this case report, which also confirms that diagnostic tests designed for the identification of BVDV infection in cattle can be used effectively to identify BVDV in alpaca. Sources and manufacturers a. Dako Cytomation Inc, Mississauga, Onatario, Canada. b. Dr. E. Dubovi, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY. c. EnVision HRP, DAKO Cytomation, Mississauga, Onatario, Canada. d. Vector Laboratories Canada Inc., Burlington, Onatario, Canada. e. IDEXX Laboratories, Westbrook, ME. References 1. Belknap EB, Collins JK, Larsen RS, Conrad KP: 2000, Bovine viral diarrhea in New World camelids. J Vet Diagn Invest 12: Carman S, van Dreumel T, Ridpath J, et al.: 1998, Severe acute bovine viral diarrhea in Ontario J Vet Diagn Invest 10: Deregt D, Bolin SR, van den Hurk J, et al: 1998, Mapping of a type 1-specific and type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants. Virus Res. 57: Deregt D, Carman PS, Clark RM, et al: 2002, A comparison of polymerase chain reaction with and without RNA extraction and virus isolation for detection of bovine viral diarrhea virus in young calves. J Vet Diagn Invest. 14: Goyal SM, Bouljihad M, Haugerud S, Ridpath JF: 2002, Isolation of bovine viral diarrhea virus from an alpaca. J Vet Diagn Invest 14: Grooms DL: 2004, Reproductive consequences of infection with bovine viral diarrhea virus. In: Veterinary Clinics North America: food animal practice, ed. Brock KV, Vol. 20, pp W. B. Saunders Company, Philadelphia, PA. 7. Haines DM, Clark EG, Dubovi EJ: 1992, Monoclonal antibodybased immunohistochemical detection of bovine viral diarrhea virus in formalin-fixed, paraffin-embedded tissues. Vet Pathol 29: Mattson DE: 1994, Viral diseases. In: Veterinary Clinics North America: food animal practice: update on llama medicine: viral diseases, ed. Johnson LW, Vol. 10, pp W. B. Saunders Company, Philadelphia, PA. 9. Nettleton PF, Gilray JA, Russo P, Dlissi E: 1998, Border disease of sheep and goats. Vet Res 29: Njaa BL, Clark EG, Janzen E, et al: 2000, Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens. J Vet Diagn Invest 12: Rivera H, Madewell BR, Ameghino E: 1987, Serological survey of viral antibodies in the Peruvian alpaca (Lama pacos). Am J Vet Res 48: Wentz PA, Belknap EB, Brock KV, et al: 2003, Evaluation of bovine viral diarrhea virus in New World camelids. J Am Vet Med Assoc 223: