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

BVDV in Alpacas

Clayton L. Kelling

ProfessorDepartment of Veterinary

and Biomedical Sciences

University of Nebraska-Lincoln

BVDV in Alpacas

- Prevalence

- Infections - Fetal- Postnatal

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

Collaborators

University of Nebraska: Christina L. Topliff David R.

Smith Sharon L. Clowser David J. SteffenJamie N. Henningson Kent M. EskridgeGary 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

Objective

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

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)

Distribution of 562 eligible alpaca herds

12

US States-www.50states.com

Distribution of 250 selected alpaca herds


1


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

Results: Seropositive herds

Herds with seropositive crias 16/63 (25.4%)

95% Confidence interval of 15.3% - 37.9%

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

Basis for herd BVDV seropositive status

BVDV Seropositive Herds

Colostrum

- DamPI exposure within herd

Off farm exposure - Supplemental

SUMMARY Case Studies of 16 Seropositive HerdsNo.Of Herds BVDV Antibody Source

6 Dam prior BVDV infection

6 Colostrum supplements(4 bovine, 1 goat, 1 both)

4 BVDV-infected herd


BVDV Seropositive Herds (n=6)

Colostrum

- DamPrior off farm exposure

Herd No. 10 BVDV Seropositive Seropositive Dam Prior Infection

Cria Bovine Antibody TiterNo. Age Colostrum BVDV 1 BVDV21 129d NO <2 <22 164d NO 8 <23 30d NO <2 <24 39d NO <2 <25 171d NO NA <26 23d NO <2 <2

DamNo.2 256 32

Herd No. 16 BVDV Seropositive Seropositive Dam Prior Infection

Cria Bovine Antibody Titer

No. Age Colostrum BVDV 1 BVDV2

1 154d NO <2 <22 166d NO <2 <23 364d NO <2 <24 183d NO 8 4

DamNo.4 256

64



Colostrum - Supplemental

Herd No. 7 BVDV Seropositive: Bovine Colostrum Fed to Crias

Cria Bovine Antibody TiterNo. Age Colostrum BVDV 1 BVDV21 207d NO <2 <22 11d YES 8 23 61d NO <2 <24 1d YES 4 4

Tested Dams 2 and 4: <2 <2

Herd No. 14 BVDV Seropositive: Bovine Colostrum Fed to

Cria

Cria Bovine Antibody Titer

No. Age Colostrum BVDV 1 BVDV2

1 60d No <2 <22 203d No <2 <23 37d Yes 32 164 61d No <2 <25 57d No <2 <2

DamNo.3 <2 <2



Colostrum

- DamPI exposure within

herd

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)

Herd No. 4 BVDV Seropositive: PI Cria

Cria Bovine Antibody TiterNo. Age Colostrum BVDV 1 BVDV21 1d NO ≥256 322 1d NO <2 <23 25d NO <2 <24 3d NO ≥256 16

Herd No. 13 BVDV Seropositive: PI Cria

Cria Bovine Antibody TiterNo. Age Colostrum BVDV 1

BVDV21 167d NO ≥ 256 ≥ 2562 131d NO ≥ 256 ≥ 1283 141d NO ≥ 256 ≥ 2564 132d NO 16 25 84d NO 64 8

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)

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

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.

BVDV Infected Herds

Herds with PI crias: 4/63 (6.4%)

95% confidence interval of 1.7% - 15.5%

Beef cattle herds with PI cattle: 3% to 4% (Wittum et al, 2001)

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.

The farms that had PI crias suffered severe economic losses due to:

- abortions, birth of weak crias that died

- expenses associated with diagnostic/treatmentregimens

- loss of sales

PI Crias from the BVDV infected herds

Objectives

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

Materials and Methods

– 10 PI alpacas– 5 PI calves– Gross, microscopic, and IHC– Scored antigen deposition

Cell types

Most prominent:

Neurons, endothelial cells, macrophages

Epithelial: Prominent in follicular epithelium, renal tubular epithelium and other endocrine and glandular cells

Kidney Salivary gland

Pancreas Thyroid

IHC. PI Ear Notch

Alpaca

PI alpaca vs. PI calf

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

Prescapular Lymph NodeAlpaca

Calf: Score 4

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

Conclusions

Fetal BVDV infections:

AbortionsPI crias

Acute BVDV infections in alpacas

Consequences of postnatal infections of alpacas?


Experimental acute BVDV infections

Alpacas vs. cattle

BVDV NY-1

BVDV CO-06

Acute BVDV Infection in Alpacas and Cattle

96

98

100

102

104

106

108

-1 0 1 2 3 4 5 6 7 8 9

Days Post-infection

Rec

tal

Tem

per

atu

re (o

F)

Alpaca

Cattle

Lymphocytes

0

1

2

3

4

5

6

7

0 1 2 3 4 5 6 7 8 9

Days Post-infection

Lym

ph

ocy

tes

(x 1

03 /ul)

CO-06 Alpaca

NY-1 Cattle

NY-1 Alpaca

Alpaca GALT. IHC

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


Summary: Consequences of postnatal infections of alpacas:

Systemic infection - Marked leukopenia &

lymphopenia

- Very mild lymphodepletion - Antigen deposition


Consequences of postnatal BVDV infections of alpacas:

Alpacas are susceptible

but are differences compared to calves

Comparative permissiveness of

alpaca and bovine cells to BVDV infections

BVDV NY-1 and CO-06 in alpaca and bovine cells

4

1 2 3

5 6

BT

AT

(-) NY-1 CO-06

Alpaca (CO-06) and bovine BVDV 1 (NY-1): alpaca and bovine cells

NY-1 vs. CO-06: TCID50

0

1

2

3

4

5

6

7

8

9

0 1.5 3 6 9 12 18 24 48 72

Hours P.I.

log

TC

ID5

0

BVDV2: Alpaca vs. bovine cells

4

1 2 3

5 6

BT

AT

(-) 890 7937

BVDV2: Alpaca vs. bovine cells

890 vs. 7937: TCID50

0

1

2

3

4

5

6

7

8

9

0 1.5 3 6 9 12 18 24 48 72

Hours P.I.

log

TC

ID5

0

Alpaca cells: BVDV1 vs. BVDV2

0

1

2

3

4

5

6

0 1.5 3 6 9 12 18 24 48 72

Hours P.I.

log

TC

ID50

BVDV NY-1 A

BVDV CO-06 A

BVDV 890 A

BVDV 7937 A

Bovine cells: Alpaca BVDV vs. Bovine BVDV

0

1

2

3

4

5

6

7

8

9

0 1.5 3 6 9 12 18 24 48 72

Hours P.I.

log

TC

ID50

BVDV 890 B

BVDV 7937 B

BVDV NY-1 B

BVDV CO-06 B

Comparative permissiveness of alpaca and bovine cells to BVDV infections

Alpaca cells less permissive than bovine cells to BVDV infections

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

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

(-) NY-1 CO-06 Poly I:C

Re

ad

ing

(R

LU

)

Inhibition of IFN synthesis by NY-1 and

Co-06 BVDV infection of AT and BT cells

0

200000

400000

600000

800000

1000000

1200000

(-) NY-1 (- PolyI:C)

NY-1 (+ PolyI:C)

CO-06 (-PolyI:C)

CO-06 (+ PolyI:C)

Poly I:C (- PolyI:C)

Poly I:C (+Poly I:C)

Re

ad

ing

(R

LU

)

Conclusions

Elevated IFN responses in BVDV infected alpaca cellscorrelates with:

–Reduced permissiveness of AT cells.

Conclusions

Elevated IFN responses in BVDV infected alpaca cellscorrelates with:

– Reduced permissiveness of AT cells.

– Mild pathologic effects of BVDV in lymphatic organs of alpacas infected postnatally.

Clinical Relevance

BVDV is important to the US alpaca industry

Clinical Relevance


Herd prevalence of 6.4%.

95% confidence interval of 1.7% - 15.5%

Clinical Relevance



BVDV is pathogenic in alpacas

Clinical Relevance




- fetal infections: PI, abortions

-

Clinical Relevance




- fetal infections: PI, abortions

- postnatal infections

Clinical Relevance



BVDV is pathogenic in alpacas- fetal infections: PI,

abortions- postnatal infections

Risk factors for BVDV exposure: Movement and commingling with unknown BVDV status

Clinical Relevance





Risk factors for BVDV exposure: Movement and commingling

Determine BVDV PI status (PCR tests) of alpacas before movement

Clinical Relevance





Risk factors for BVDV exposure: Movement and commingling

Determine BVDV PI status (PCR tests) of alpacas before movement

Acknowledgments

– Funding:

Mid-Atlantic Alpaca Association through the

Alpaca Research Foundation

University of Nebraska-LincolnAgricultural Research

DivisionInstitute of Agriculture and

Natural Resources

IHC and Histopathology Scales

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– 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– 0: None – 1: Mild– 2: Moderate– 3: Severe

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

Interferon response in bovine and alpaca cells

NCL1 Luc ISRE cells

Gil et al., 2004


Luciferase


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


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

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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