1. BVDV in Alpacas Clayton L. Kelling Professor Department of Veterinary
and Biomedical Sciences
University of Nebraska-Lincoln

2. Prevalence Infections Fetal Postnatal
BVDV in Alpacas
Prevalence
Infections
Fetal
Postnatal

3. BVDV in North American Alpaca Herds
- Multiple confirmed cases of PI alpacas and reproductive failure in N. America
- Prevalence unknown
- ARF RFP: determine the prevalence of BVDV

4. Collaborators University of Nebraska:
Christina L. Topliff David R. Smith
Sharon L. Clowser David J. Steffen
Jamie N. Henningson Kent M. Eskridge
Gary P. Rupp Bruce W. Brodersen
Clayton L. Kelling
Daniela Bedenice Tufts University
Robert J. Callan Colorado State University
Carlos Reggiardo University of Arizona
Kathy L. Kurth University of Wisconsin

5. Objective
to determine the current prevalence of BVDV-infected US alpaca herds

6. Approach Tested 5 crias from a randomly selected sample of herds for:
BVDV antibodies
BVDV RNA
BVDV
US alpaca herds with 12 or more registered females
250/562 eligible herds (AOBA Directory)

7. Distribution of 562 eligible alpaca herds12
US States-

8. Distribution of 250 selected alpaca herds1
US States-

9. Sixty-three alpaca herds located in 26 states participated in the study
US States-

10. Results: Seropositive herds
Herds with seropositive crias 16/63 (25.4%)
95% Confidence interval of 15.3% %

11. Case Studies of the 16 Herds with Seropositive Crias
- Individually contacted
- Detailed information
- Scheduled follow-up testing of the dams
- Whole herd RT-PCR testing:
confirmed BVDV-free status
- Seropositive dams in BVDV-free herd: indicated prior BVDV exposure

12. Basis for herd BVDV seropositive status
BVDV Seropositive Herds
Colostrum
- Dam
PI exposure within herd
Off farm exposure
- Supplemental

13. SUMMARY Case Studies of 16 Seropositive Herds
No.
Of Herds BVDV Antibody Source
6 Dam prior BVDV infection
6 Colostrum supplements
(4 bovine, 1 goat, 1 both)
4 BVDV-infected herd

14. Basis for herd BVDV seropositive status
BVDV Seropositive Herds (n=6)
Colostrum
- Dam
Prior off farm exposure

15. Herd No. 10 BVDV Seropositive Seropositive Dam Prior Infection
Cria Bovine Antibody Titer
No. Age Colostrum BVDV 1 BVDV2
1 129d NO <2 <2
2 164d NO <2
d NO < <2
d NO < <2
5 171d NO NA <2
d NO < <2
Dam
No.

16. Herd No. 16 BVDV Seropositive Seropositive Dam Prior Infection
Cria Bovine Antibody Titer
No Age Colostrum BVDV BVDV2
d NO < <2
d NO < <2
d NO < <2
d NO
Dam
No.

17. Basis for herd BVDV seropositive status
BVDV Seropositive Herds (n=6)
Colostrum
- Supplemental

18. Herd No. 7 BVDV Seropositive: Bovine Colostrum Fed to Crias
Cria Bovine Antibody Titer
No. Age Colostrum BVDV 1 BVDV2
1 207d NO <2 <2
2 11d YES
3 61d NO <2 <2
d YES
Tested Dams 2 and 4: < <2

19. Herd No. 14 BVDV Seropositive: Bovine Colostrum Fed to Cria
Cria Bovine Antibody Titer
No Age Colostrum BVDV 1 BVDV2
d No <2 <2
d No <2 <2
d Yes
d No <2 <2
d No <2 <2
Dam
No.
< <2

20. Basis for herd BVDV seropositive status
BVDV Seropositive Herds (n=4)
Colostrum
- Dam
PI exposure within herd

21. Results: Herds with PI crias
Seropositive herds with PI crias: 4/63
- One herd detected by testing samples
. Three herds recently had PI cria
(Case studies)

22. Herd No. 4 BVDV Seropositive: PI Cria
Cria Bovine Antibody Titer
No. Age Colostrum BVDV 1 BVDV2
d NO ≥
d NO <2 <2
d NO <2 <2
d NO ≥

23. Herd No. 13 BVDV Seropositive: PI Cria
Cria Bovine Antibody Titer
No. Age Colostrum BVDV 1 BVDV2
1 167d NO ≥ ≥ 256
2 131d NO ≥ ≥ 128
3 141d NO ≥ ≥ 256
4 132d NO
d NO

24. Source of infection in 3 of the 4 infected herds was linked
- Based on genetic homologies of viruses
- PCR amplified BVDV 5’ untranslated region (UTR) PCR products from PI animals
- Sequencing
- 283 base pair (bp) fragment of the 5’ UTR (nucleotides 107 and 389)

25. Source of infection in 3 herds with PI crias were linked:
- High genetic homologies (99.2 and 99.6%)
confirmed the common source of infection
- Gestating females in 2 herds contacted a PI cria in the same herd

26. Source of BVDV infection in the fourth herd (No. 15) was unique
- Nucleotide identity:
- 91.1% with Herd Nos, 4, 11 and 13
- Exposed to a breeding female with a PI
cria at side from another farm.

27. BVDV Infected Herds Herds with PI crias: 4/63 (6.4%)
95% confidence interval of 1.7% %
Beef cattle herds with PI cattle: 3% to 4%
(Wittum et al, 2001)

28. Discussion
The prevalence (6.3%) of BVDV-infected alpaca
herds with PI crias was relatively high …..
……attributable to transporting female alpacas with PI crias at side to various sites for breeding.

29. The farms that had PI crias suffered severe economic losses due to:
- abortions, birth of weak crias that died
- expenses associated with diagnostic/treatment
regimens
- loss of sales

30. PI Crias from the BVDV infected herds
Npro 30

31. Objectives
Determine the extent of lesions and antigen distribution in PI alpacas and compare to PI calves

32. Materials and Methods 10 PI alpacas 5 PI calves
Gross, microscopic, and IHC
Scored antigen deposition

33. Cell types Neurons, endothelial cells, macrophages Most prominent:
Epithelial: Prominent in follicular epithelium, renal tubular epithelium and other endocrine and glandular cells

34. Kidney Salivary gland
Thyroid

35. IHC. PI Ear Notch Alpaca
Figure 5: Photomicrograph of cerebrum. A) Alpaca D. Scattered deposition of BVDV antigen is present. Immunohistochemical stain; bar=50 um. B) Bovine B. Intense almost diffuse deposition of BVDV antigen is evident. Immunohistochemical stain; bar=50 um. 35

36. PI alpaca vs. PI calf

37. __ Distribution Intensity Associated Lesions
Distribution of BVDV antigen in lymphoid organs of PI alpacas and calves
BVDV Antigen
__ Distribution Intensity Associated Lesions
PI calves Wide Marked None
PI alpacas Wide Moderate None

38. Prescapular Lymph Node
Alpaca
Calf: Score 4
Figure 3: Photomicrograph of prescapular lymph node. A) Alpaca B. Weak deposition of BVDV antigen occurred. Immunohistochemical stain; bar=50 um. B) Bovine B. Marked deposition of BVDV antigen is evident. Immunohistochemical stain; bar=25 um.

39. Graph displaying mean antigen deposition scores of selected organs systems between alpacas and calves.

40. Conclusions PI alpacas:
Widespread distribution of BVDV antigen affecting multiple organ systems and cell types
Widespread epithelial staining suggests nasal, oral, and urinary fluids may be infectious

41. Fetal BVDV infections: Abortions PI crias
Conclusions
Fetal BVDV infections:
Abortions
PI crias
Npro 41

42. Acute BVDV infections in alpacas
Consequences of postnatal infections of alpacas?

43. Acute BVDV infections in alpacas
Experimental acute BVDV infections
Alpacas vs. cattle
BVDV NY-1
BVDV CO-06

46. Alpaca GALT. IHC

47. Distribution Associated lesions
Distribution of BVDV antigen in lymphoid tissues of acutely-infected alpacas and calves
BVDV Antigen
Distribution Associated lesions
Calves PP,MLN,Thy Marked
Alpacas (C3, PP, Colon) Very Mild
Variable
***Preliminary findings
One alpaca had widely disseminated deposition of antigen in lymphoid organs throughout body, while in 4 other alpacas antigen was limited to the lymphoid tissues of C3 or was not detected at all.

48. Acute BVDV infections in alpacas
Summary: Consequences of postnatal infections of alpacas:
Systemic infection
- Marked leukopenia & lymphopenia
- Very mild lymphodepletion
- Antigen deposition

49. Acute BVDV infections in alpacas
Consequences of postnatal BVDV infections of alpacas:
Alpacas are susceptible
but are differences compared to calves

50. Comparative permissiveness of alpaca and bovine cells to BVDV infections

51. BVDV NY-1 and CO-06 in alpaca and bovine cells
(-)
NY-1
CO-06 1 2 3 BT
1=BT negative
2=BT NY-1
3=BT CO-06
4=AT negative
5= AT NY-1
6=AT CO-06 4 5 6 AT

52. Alpaca (CO-06) and bovine BVDV 1 (NY-1): alpaca and bovine cells
NY-1 vs. CO-06: TCID50
TCID50

53. BVDV2: Alpaca vs. bovine cells
(-)
890
7937 1 2 3 BT
1=BT negative
2=BT 890
3=BT 7937
4=AT negative
5= AT 890
6=AT 7937 4 5 6 AT

54. BVDV2: Alpaca vs. bovine cells
890 vs. 7937: TCID50

55. Alpaca cells: BVDV1 vs. BVDV2

56. Bovine cells: Alpaca BVDV vs. Bovine BVDV

57. Alpaca cells less permissive than bovine cells to BVDV infections
Comparative permissiveness of alpaca and bovine cells to BVDV infections
Alpaca cells less permissive than bovine cells to BVDV infections

58. IFN I responses of alpaca testicular (AT) and bovine turbinate (BT) cells infected with BVDV CO-06 or NY-1.

59. Inhibition of IFN synthesis by NY-1 and Co-06 BVDV infection of AT and BT cells

60. Elevated IFN responses in BVDV infected alpaca cells
Conclusions
Elevated IFN responses in BVDV infected alpaca cells
correlates with:
Reduced permissiveness of AT cells.

61. Elevated IFN responses in BVDV infected alpaca cells
Conclusions
Elevated IFN responses in BVDV infected alpaca cells
correlates with:
Reduced permissiveness of AT cells.
Mild pathologic effects of BVDV in lymphatic organs of alpacas infected postnatally.

62. BVDV is important to the US alpaca industry
Clinical Relevance
BVDV is important to the US alpaca industry

63. BVDV is important to the US alpaca industry
Clinical Relevance
BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
95% confidence interval of 1.7% %

64. BVDV is important to the US alpaca industry
Clinical Relevance
BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas

65. Clinical Relevance
BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas
- fetal infections: PI, abortions -

66. Clinical Relevance BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas
- fetal infections: PI, abortions
- postnatal infections

67. Clinical Relevance BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas
- fetal infections: PI, abortions
- postnatal infections
Risk factors for BVDV exposure:
Movement and commingling
with unknown BVDV status

68. Clinical Relevance BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas
- fetal infections: PI, abortions
- postnatal infections
Risk factors for BVDV exposure:
Movement and commingling
Determine BVDV PI status (PCR tests) of alpacas before movement

69. Clinical Relevance BVDV is important to the US alpaca industry
Herd prevalence of 6.4%.
BVDV is pathogenic in alpacas
- fetal infections: PI, abortions
- postnatal infections
Risk factors for BVDV exposure:
Movement and commingling
Determine BVDV PI status (PCR tests) of alpacas before movement

70. Acknowledgments Funding: Mid-Atlantic Alpaca Association through the
Alpaca Research Foundation
University of Nebraska-Lincoln
Agricultural Research Division
Institute of Agriculture and Natural Resources

72. IHC and Histopathology Scales
0: None
1: Positive single or small group
2: Scattered staining, clearly positive
3: Widespread, multifocal, many
4: Intense widespread, diffuse, most follicles
Lymphocytolysis
1: Scattered cells (normal)
2: Many cells, one or more follicles affected
3: Moderate (25-50%)
4: Widespread, most follicles (>50%)
Lymphoid depletion
0: None
1: 1-10%
2: 10-25%
3: 25-50%
4>50%
Inflammation and Necrosis
1: Mild
2: Moderate
3: Severe

73. Photomicrograph of sections of Peyer’s patches of calf acutely infected with BVDV 890, 9 days PI.

74. Interferon response in bovine and alpaca cells
NCL1 Luc ISRE cells
Upon activation of associated gene (ISRE responds to interferon) luciferin is made
Assay buffer adds other component to create a chemoluminescent effect (Promega) that can be measured by a luminometer
Gil et al., 2004

75. Interferon response in bovine and alpaca cells
Luciferase
ATP plus magnesium to oxidize luciferin

76. Interferon response in bovine and alpaca cells
Interferon production assay
BT or AT cells infected at MOI of 10, mock infection control with uninfected growth media
Incubated 24 hours
BVDV inactivated with changes in pH
Stimulation of NCL1 ISRE-Luc cells
Cells grown on 12 well plate and overlaid with test sample
Luciferase assay
Cells lysed and fluorescence measured
NCL1 Luc ISRE cells express luciferase with an interferon responsive element

77. Interferon response in bovine and alpaca cells
Interferon inhibition assay
AT and BT cells infected at an m.o.i. of 1, incubated 48 hours, then stimulated with poly I:C, a dsRNA analogue
Assayed for interferon response using reporter cell line