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BVDV in Alpacas Clayton L. Kelling Clayton L. Kelling Professor Professor Department of Veterinary and Biomedical Sciences and Biomedical Sciences University.

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Presentation on theme: "BVDV in Alpacas Clayton L. Kelling Clayton L. Kelling Professor Professor Department of Veterinary and Biomedical Sciences and Biomedical Sciences University."— Presentation transcript:

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

2 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 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 antibodies BVDV RNA BVDV RNA BVDV BVDV US alpaca herds with 12 or more registered females 250/562 eligible herds (AOBA Directory)

7 Distribution of 562 eligible alpaca herds 12 US States-www.50states.com

8 Distribution of 250 selected alpaca herds US States-www.50states.com 1

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

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 Colostrum - Dam PI exposure within herd Off farm exposure Off farm exposure - Supplemental - Supplemental

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

14 Basis for herd BVDV seropositive status BVDV Seropositive Herds (n=6) Colostrum 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 BVDV d NO <2 < d NO 8 <2 3 30d NO <2 <2 4 39d NO <2 < d NO NA <2 6 23d NO <2 <2 DamNo

16 Herd No. 16 BVDV Seropositive Seropositive Dam Prior Infection Cria Bovine Antibody Titer No. Age Colostrum BVDV 1 BVDV d NO <2 < d NO <2 < d NO <2 < d NO 8 4 DamNo

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

18 Herd No. 7 BVDV Seropositive: Bovine Colostrum Fed to Crias Cria Bovine Antibody Titer No. Age Colostrum BVDV 1 BVDV2 1207d NO <2<2 2 11d YES d NO <2<2 4 1d YES 4 4 Tested Dams 2 and 4: <2 <2

19 Herd No. 14 BVDV Seropositive: Bovine Colostrum Fed to Cria Cria Bovine Antibody Titer No. Age Colostrum BVDV 1 BVDV2 1 60d No <2 < d No <2 <2 3 37d Yes d No <2 <2 5 57d No <2 <2 DamNo. 3 <2 <2

20 Basis for herd BVDV seropositive status BVDV Seropositive Herds (n=4) Colostrum 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 1 1d NO ≥ d NO <2 <2 3 25d NO <2 <2 4 3d NO ≥256 16

23 Herd No. 13 BVDV Seropositive: PI Cria Cria Bovine Antibody Titer No. Age Colostrum BVDV 1 BVDV d NO ≥ 256 ≥ d NO ≥ 256 ≥ d NO ≥ 256 ≥ d NO d NO 64 8

24 Source of infection in 3 of the 4 infected herds was linked - Based on genetic homologies of viruses - Based on genetic homologies of viruses - PCR amplified BVDV 5 ’ untranslated region (UTR) PCR products from PI animals - Sequencing 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: % 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

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 Most prominent: Neurons, endothelial cells, macrophages 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 IHC. PI Ear Notch Alpaca

36 PI alpaca vs. PI calf

37 Distribution of BVDV antigen in lymphoid organs of PI alpacas and calves BVDV Antigen __ Distribution Intensity Associated Lesions PI calves Wide MarkedNone PI alpacas Wide ModerateNone

38 Prescapular Lymph Node Alpaca Calf: Score 4

39

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 Conclusions Fetal BVDV infections: Abortions PI crias

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 NY-1 BVDV CO-06 BVDV CO-06

44

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46 Alpaca GALT. IHC

47 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

48 Acute BVDV infections in alpacas Summary: Consequences of postnatal infections of alpacas: Systemic infection - Marked leukopenia & lymphopenia - Marked leukopenia & lymphopenia - Very mild lymphodepletion - Very mild lymphodepletion - Antigen deposition - 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 BT AT (-)NY-1CO-06

52 Alpaca (CO-06) and bovine BVDV 1 (NY-1): alpaca and bovine cells NY-1 vs. CO-06: TCID 50

53 BVDV2: Alpaca vs. bovine cells BT AT (-)

54 BVDV2: Alpaca vs. bovine cells 890 vs. 7937: TCID 50

55 Alpaca cells: BVDV1 vs. BVDV2

56 Bovine cells: Alpaca BVDV vs. Bovine BVDV

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

61 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 Clinical Relevance BVDV is important to the US alpaca industry

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

64 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 Movement and commingling with unknown BVDV status 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 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 Movement and commingling Determine BVDV PI status (PCR tests) of alpacas before movement

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

71

72 IHC and Histopathology Scales IHC IHC –0: None –1: Positive single or small group –2: Scattered staining, clearly positive –3: Widespread, multifocal, many –4: Intense widespread, diffuse, most follicles Lymphocytolysis Lymphocytolysis –1: Scattered cells (normal) –2: Many cells, one or more follicles affected –3: Moderate (25-50%) –4: Widespread, most follicles (>50%) Lymphoid depletion Lymphoid depletion –0: None –1: 1-10% –2: 10-25% –3: 25-50% –4>50% Inflammation and Necrosis Inflammation and Necrosis –0: None –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 Gil et al., 2004

75 Interferon response in bovine and alpaca cells Luciferase

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


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