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SEROLOGICAL EVALUATION OF SENTINEL CALVES IN A

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BVDV ERADICATION PROGRAM

presented by
Erik Matthew Corbett

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degree in Large Animal Clinical Sciences

 

 

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SEROLOGICAL EVALUATION OF SENTINEL CALVES IN A BVDV
ERADICATION PROGRAM

By

Erik Matthew Corbett

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Large Animal Clinical Sciences

2010

ABSTRACT

SEROLOGICAL EVALUATION OF SENTINEL CALVES IN A BVDV
ERADICATION PROGRAM

By

Erik Matthew Corbett

The identification and removal of persistently infected (PI) cattle is critical to
eradicating bovine viral diarrhea virus (BVDV). Serological evaluation of small groups
of young unvaccinated calves has been proposed as an alternative method for identifying
herds with cattle persistently infected (PI) with BVDV. The objective of the study
reported here was to evaluate the application of sentinel serology as an on-going herd
monitoring tool in herds enrolled in a regional BVDV eradication project.

Serum samples were collected from 47 management groups from 36 herds. Virus
neutralization (VN) antibody titers to type 1 and type 2 BVDV were determined from
five non-vaccinated calves > 6 months of age in each management group. A management
group was considered to be BVDV positive if 2 of 5 calves had VN titers greater than 2
128. Skin samples from all calves in all herds were analyzed using reverse transcriptase
polymerase chain reaction (RT-PCR) to identify PI animals from any infected herds.

PI cattle were identified in one herd with one management group. In that herd, 3
sentinel calves had VN antibody titers 2128. All other management groups were
negative for BVDV. The K value for agreement between sentinel serology and RT-PCR
was 1.0 (95% CI 1.0 to 1.0). This study further supports the use of sentinel serology for
identifying cattle herds With BVDV and its potential use as a tool in BVDV eradication

programs.

DEDICATION

It is my honor to dedicate this work to my parents
Keith and Adrienne Corbett.
Thank you .for all of your love and support throughout the years,
especially during the time when I was searching for the
right career in veterinary medicine.

ACKNOWLEDGMENTS

I would like to thanks my graduate committee members, Drs. Dan Grooms, Steve
Bolin, and Ben Bartlett, for their guidance and support in completing this project. I
would especially like to the Dr. Grooms for providing me with the opportunity to return
to graduate school and for putting up with me the past year and a half. You have been a
great mentor and friend to me and I will attempt to carry all that I’ve learned into my

future endeavors.

TABLE OF CONTENTS

 

 

 

 

 

LIST OF TABLES ...... -- - - vvi
LIST OF FIGURES _ -- - -- -- - u -- -- -- - - -- -- - - - - .......... viii
KEY TO ABREVIATIONS -- --------- - - - - - - - -..viiiii
INTRODUCTION . . - - - c - - -_ - ......... ‘ 1
LITERATURE REVIEW .................. 3

SEROLOGICAL EVALUATION OF SENTINEL CALVES IN A BVDV
ERADICATION PROGRAM - - 41

 

IMMUNIZATION OF CALVES WITH A MODIFIED-LIVE BOVINE VIRAL
DIARRHEA VIRUS VACCINE: EVALUATION OF BOVINE VIRAL
DIARRHEA VIRUS IN CLINICAL SAMPLES POST VACCCINATION ............. 58

SUMMARY - . - - 75

 

LIST OF TABLES

CHAPTER 1

TABLE 1.1. Prevalence of BVDV PI cattle in various studies-_----...

 

TABLE 1.2. BVDV testing strategies

 

TABLE 1.3. BVDV vaccine attributes .....

 

 

TABLE 1.4. BVDV vaccination strategies

CHAPTER 2

TABLE 2.1. Herd demographic information and results of herd

 

testing using calf ear notching and sentine serology

TABLE 2.2. Sensitivity and specificity of serological evaluation of
VN antibodies against BVDV using different diagnostic criteria and
when compared with pooled RT-PCR of skin samples

 

CHAPTER 3

TABLE 3.1. BVDV biotypes isolated in Experiment 2: Days 7-14

 

TABLE 3.2. Virus Neutralization results at day 0 and day 18 post
vaccination with a commercial modified-live BVDV vaccine

 

vi

12

23

27

28

52

54

-70

-- 71

LIST OF FIGURES

CHAPTER 3

FIGURE 3.1. Phylogenetic relationships of 14 BVDV isolates to
genotype 1 (Vaccine Type 1) and genotype 2 (Vaccine Type 2) BVDV
vaccine used in experiment 2.

 

vii

72

ACE

BRD

BVDV

BVD

CI

CPE

DCPAH

DMEM

EED

ELISA

IHC

MLV

RCF

PI

RT-PCR

VI

KEY TO ABREVIATIONS

Antigen Capture ELISA

Bovine Respiratory Disease

Bovine Viral Diarrhea Virus

Bovine Viral Diarrhea

Confidence Interval

Cytopathic Effects

Diagnostic Center for Population and Animal Health
Dullbecco’s Modified Eagle Medium

Early Embryonic Death

Enzyme Linked Immunosorbent Assay
Immunohistochemistry

Modified Live Virus

Relative Centrifugal Force

Persistently Infected or Persistent Infection

Reverse Transcriptase - Polymerase Chain Reaction
Untranslated Region

Virus Isolation

Virus Neutralization

viii

INTRODUCTION

Bovine viral diarrhea virus (BVDV) is one of the most economically devastating
diseases of cattle, affecting animals throughout the world. Control of bovine viral
diarrhea virus (BVDV) has been a worldwide challenge for decades. Various clinical
manifestations can result fiom infection with BVDV, ranging from subclinical infection
to immunosuppresssion and respiratory disease to abortion and infertility. The most well
known source for transmission of BVDV are persistently infected (PI) cattle. Persistent
infection occurs when calves are exposed to BVDV in utero before the fetal immune
system is developed, thus allowing the developing fetus to become immunotolerant to the
virus. PI cattle continuously shed large amounts of virus into the environment and are an
important source of virus transmission within and between herds.

In 2007, the Michigan Upper Peninsula BVDV Eradication Program was
launched. The purpose of this project is to eradicate BVDV from a geographic area and,
in doing so, identify benefits and obstacles of a BVDV eradication program and
demonstrate a feasible model that may be adopted by other parts of the US. Key
components of the program include identification and removal of cattle persistently
infected with BVDV, institution of a planned biosecurity program, and appropriate
BVDV vaccination. Whole herd testing to identify cattle persistently infected with
BVDV is a major investment of both time and money for producers. Therefore,
development of more efficient herd testing strategies would be beneficial. Serological
evaluation of small groups of young unvaccinated calves has been proposed as an

alternative method for identifying herds with cattle persistently infected (PI) with BVDV.

The objective of the study reported here was to evaluate the application of
sentinel serology as an on-going herd monitoring tool in herds enrolled in a regional
BVDV eradication project. The goal of sentinel testing is to provide an effective herd

screening tool that can be used in follow-up surveillance in an eradication program.

CHAPTER 1
LITERATURE REVIEW

Introduction

Bovine viral diarrhea virus (BVDV) is an important pathogen affecting cattle
worldwide and is one of the most economically devastating diseases of the cattle
industry. The initial clinical disease, named Bovine Viral Diarrhea (BVD), was first

described in the United States in 1946 and was associated with gastroenteritis and severe
diarrhea, with erosive lesions of the digestive tract.1 The disease was associated with high

morbidity and low mortality rates. Since then the virus has been isolated throughout the
world from a variety of animals including cattle, sheep, llamas, and deer. Infection with
BVDV can manifest itself in several different syndromes including diarrhea, respiratory
disease, immunosuppression, subclinical infection, and reproductive failure. Causes of
reproductive failure associated with BVDV include infertility, early embryonic death
(BED), abortion, and congenital defects. The virus can also impair the immunity of
infected animals which can aggravate other diseases or increase the susceptibility to other
disease complexes such as bronchopneumonia, diarrhea, and mastitis. One of the more
unique characteristics of BVDV is its ability to create persistently infected (PI) cattle as a
consequence of in utero infection by noncytopathic BVDV between days 40 and 125 of
gestation. Infected fetuses that survive until birth are born immunotolerant to the specific
exposing viral strain and are lifelong shedders of the virus. PI cattle are the principle

reservoir of BVDV and serve as the primary source of viral spread.

The following review will review in firrther detail what is known about BVDV:
the types of infection, prevalence and incidence of the disease, virus transmission,

diagnostic strategies, control and prevention, and eradication.

The Virus

Bovine viral diarrhea virus is a positive-sense, single-stranded RNA virus. It
belongs to the genus Pestivirus in the F laviviridae family. BVDV isolates are fitrther
broken down by their genotype and biotype.

In 1993, there were sudden deaths among adult dairy cattle in Canada and parts of

the northeastern U.S.2 Genetic typing found that these BVDV isolates were quite

different than the recognized strains in existence.3 With the advent of polymerase chain

reaction (PCR) in the 1990’s, it became possible to differentiate BVDV into two different
genotypes: type 1 and type 2. These genotypes were further broken down into

subgenotypes. Currently there are 11 subgenotypes of BVDV type 1 (a-k) and two

subgenotypes of BVDV type 2 (a and b).4 The most common subtypes in North America
are type 1a, 1b, and 2a.5 Type 1 strains are most commonly associated with subclinical or

mild respiratory disease.4 Type 2 isolates tend to be associated with more severe disease,
including hemorrhagic syndrome. BVDV type 2 represents up to 50% of laboratory
isolates in North America.6'9 A recent study of BVDV isolates in North America and
Australia summarized data from around the world suggesting that BVDV type 2 is rare in

countries outside of North America.10

Within each genotype of BVDV exist two biotypes. The biotype of the virus
refers to the ability of the virus to cause cell pathology in infected cell cultures.
Cytopathic virus causes cell death in vitro, while noncytopathic virus causes no visible

cell damage. Studies have shown that most BVDV field isolates are noncytopathic.7’ll

Noncytopathic stains of BVDV also tend to be more virulent.ll

Cytopathic BVDV was discovered several years after noncytopathic isolates and

it is believed that cytopathic BVDV evolved as a mutant strain of a noncytopathic virus.12

To date, only noncytopathic BVDV has been isolated from P1 cattle. I 3’14 Since PI cattle

typically represent the primary reservoir of BVDV within the population, it would
suggest that noncytopathic BVDV is the natural form of the virus, while cytopathic

BVDV strains are an atypical form.

Acute Infection with BVDV
Acute BVDV infection refers to infection that occurs in cattle that are not

persistently infected. Most BVDV infections are subclinical in nature and therefore go
undetected.15 These cattle may develop a mild fever and become leukopenic prior to

developing serum-neutralizing antibodies and clearing the virus.

Clinical infection tends to occur in young cattle between six months and two
years of age. After a 5-7 day incubation period, affected cattle develop high fevers and
severe leucopenia. Clinical signs may include fever (40-41 °C), oculonasal discharge,
depression, anorexia, decreased milk production, transient watery diarrhea, and erosions

or shallow ulcerations in the oral cavity. Additional signs can occur sporadically. The

course varies from 2-3 days up to 3 weeks with high morbidity and low mortality.
Affected animals shed the virus for up to 15 days post infection.

Bovine viral diarrhea virus is an important component of the bovine respiratory
disease complex (BRD). The primary role of BVDV in BRD is likely
immunosuppression. The leukopenia that arises from infection with the virus destroys or
inhibits the normal function of white blood cells and allows for secondary bacterial
pathogens an ideal environment to establish infection.

As mentioned previously, type 2 BVDV was first isolated from disease outbreaks
in Canada and in the northeastern US. in 1993.2’16 Infections with type 2 BVDV during

these outbreaks resulted in mortality rates as high as 20-25% in individual herds. Clinical
signs include marked thrombocytopenia, bloody diarrhea, epistaxis, hyphema, bleeding
from injection sites, pyrexia, pneumonia, and severe hemorrhage throughout the body.

This disease manifestation is referred to as hemorrhagic syndrome.

Fetal Infection with BVDV

Fetal infection occurs when a pregnant dam becomes acutely infected with BVDV
or when a persistently infected dam becomes pregnant. The result of BVDV fetal
infection depends on the stage of gestation in which the fetus is infected, as well as the

virulence of the virus.

B VD V infection prior to 50 days of gestation

Infection at the time of insemination or shortly after has been associated with low

. 17 . .
conception rates. The reason for low conception rates rs not clear but may depend on

the stage of early reproductive events and the time of infection. One reason for lower
conception rates during the first weeks of gestation is EED and resorption of the embryo.
The cow will become a repeat breeder and will return to estrus after a prolonged period of
time.

Another reason for lower conception rates could be that a transient endometritis is

8

created by the virus, resulting in inhibition of normal embryonic implantation.l In

addition, virus has been isolated in ovarian tissue following acute infection with both
19-22

cytopathic and noncytopathic BVDV.

Infections in the first 50 days are often unnoticed without carefirl monitoring at
the herd level. Increase in the number of services per conception and irregular estrus

cycles are suggestive of EED, and BVDV should be considered as a possible cause.

B VD V Median 51-] 00 davs of gestation

Fetal infection 51-100 days into gestation can result in fetal death followed by
abortion or mummification.l7’23 Several weeks or months may pass before the fetus is

expelled from the cow.

Abortions caused by BVDV can be difficult to diagnose and many aborted fetuses
associated with BVDV are negative on virus isolation.13 A major contributing factor is

the delay in fetal expulsion following BVDV infection. Because of the delay, BVDV is

often no longer present in the fetus. In addition, cows have often seroconverted by the

time of fetal expulsion , making acute and convalescent titers of little value.24

B VDV infection 101-150 days of gestation

Nervous system congenital defects are a concern following transplacental
infection between 101-150 days in gestation.17 During this period the fetus is in the final
stages of nervous system development. The inflammatory response to BVDV from the
fetal immune system can injure the developing nervous system, leading to inhibition of
cellular growth, cell differentiation, or cell lysis.13 The most common defect associated
with BVDV infection is cerebellar hypoplasia. Difficulty standing, a wide based stance,

25,26

and intention tremors are associated with cerebellar hypoplasia in calves. Other

congenital defects seen include ocular problems (blindness, retinal degeneration, and

cataracts) and skeletal defects (small malformed calves).

B VDV infection after 150 days of gestation
After approximately 150 days of gestation, immunocompetence and

organogenesis are usually complete in the developing fetus. Abortion and premature
births can occur in cases in which the infecting virus is particularly virulent.25’27
Transplacental infections with BVDV at this point often result in the birth of normal
calves seropositive to BVDV.l7 However, one study suggests that congenital infections

during the last trimester of gestation may have a negative effect on neonatal performance
and survivability, as calves born with BVDV-neutralizing antibodies were twice as likely

to experience severe illness within the first 10 months of life.28 In addition, a recent case

study of an outbreak of late term abortions and premature births associated BVDV

identified brachygnathism, thrombocytopenia, malformations of the brain and cranium,

and rare extracranial skeletal malformations.29

Persistent infection with BVDV

Persistent infection with BVDV occurs when the fetus is exposed to
noncytopathic virus between days 18 and 125 of gestation.”’17 Fetal infection during this
window is the only knoWn way of creating persistently infected cattle. It occurs during
the period when the immune system is identifying self-antigens and virus circulating is
recognized as a normal part of the fetus. The fetus becomes immunotolerant to the virus
and cannot clear the virus.30 The calf is born a lifelong carrier of the virus and is referred
to as being persistently infected or PI.

PI’s have virus in every cell of their body and shed large amounts of virus in all

excretions and secretions during its life.30’3l PI calves may appear clinically normal and

survive well into adulthood, but most are small and unthrifty.32'34 They often are culled

or die prior to becoming adults. There is no cure for PI’s.

PI cattle serve as the major source of BVDV transmission both within and

14,15,35-39

between herds. They are continuously shedding virus throughout their lives,

serving as a source of infection for naive herd mates. The few PI cattle that survive to
adulthood and give birth produce offspring that are also PI’s because the virus is present
during 50 and 125 days of gestation.40

To date, only noncytopathic strains have been isolated fi'om PI cattle and BVDV

12,17,37

immunotolerance is strain specific. PI calves will not form virus neutralizing

antibodies to the BVDV strain that they are immunotolerant to. However, it does not
preclude them from becoming infected with a heterologous BVDV strain and

subsequently developing antibodies to that strain.

Mucosal Disease

Mucosal disease occurs in P1 animals that become super-infected with a
cytopathic BVDV strain that is homologous to its immunotolerant noncytopathic strain.41

These cattle have both noncytopathic and cytopathic BVDV isolated from them, but they
typically have no viral neutralizing antibodies. The most likely origin of the cytopathic
strain is within the PI animal as a mutation of the persistent noncytopathic strain. Other

potential sources are external and included either a cytopathic field strain or modified-
live vaccine strain.13

Clinical signs of mucosa] disease include fever, depression, anorexia, weight loss,
and severe dehydration. Physical examination can reveal oral lesions on the lips, gingival
margins, tongue, and dental pad; they are often accompanied by ptyalism. Large areas of
mucosal necrosis and sloughing are present if the lesions ulcerate. Ulcerating lesions may
also be present on the vulva, teats, and interdigital areas resulting in lameness.
Intermittent to chronic diarrhea may be present. The disease is often acute with death in
3-10 days after onset of clinical signs. Postmortem examination of the gastrointestinal
tract reveals areas of necrosis and mucosal sloughing throughout (esophagus, rumen,

. . . 12,13,42
abomasums, duodenum, jejunum, rleum, cecum, and colon).

Occurrence of mucosal disease is sporadic, with mortality approaching 100% in

affected cattle.41 Outbreaks have also been reported in herds where there is a cohort of PI

10

calves. In this situation,_the virus in one PI calf mutates into a cytopathic virus, which

then infects its PI herd mates.43’44 Vaccine-associated outbreaks of mucosal disease have

also been reported, as most commercial modified-live BVDV vaccines contain cytopathic

. 4]
Vll'US.

Prevalence of BVDV Infection
BVDV infection has been found in cattle worldwide. The prevalence of BVDV
infections is often reported as the percent of PI cattle or the percent of seropositive cattle

in a population.45 It has been estimated that 4% of cow-calf herds and up to 15% of dairy

herds in the United States have persistently infected (PI) animals in them.39’46'48

Seroprevalence studies looking at BVDV antibodies have been conducted throughout the
world; with a wide range in seroprevalence (IS-89%). Herd seroprevalence can be
affected by use of vaccination and may not reflect true disease prevalence. At the

individual animal level, PI prevalence ranges from 0-1 .7% (Table l.1).32’34’39’46’47’49'53

Prevalence of PI’s can be higher in individual farms where BVDV control programs are

lacking.

11

Table 1.] Prevalence of BVDV PI cattle in various studies

 

 

 

 

 

 

 

 

 

 

 

 

Source of # of PI Total % of Total % of
Location Po ulation cattle # of PI # of PI Ref.
p cattle cattle herds herds
Nigifaléa’ (331:; Sand 54 3157 1.7% 66 4% 39
Michigan 113:3: 71 5481 1.3% 20 15% 37
Saskatchewan iii? 5 5129 0.1% 1 100% 25
Alabama,
Nebraska, 0 0
Nevada, North Beefherds 56 18,931 0.03 A. 128 4/0 31
Dakota, Ohio
Texas F322;? 6 2000 0.3% 1 100% 27
Missouri 1:33;? 3 938 0.32% 2 100% 43
Kansas 2:32? 86 21,743 0.4% 1 100% 41
““32: and Beef calves 25 45300 0.55% 30 16.7% 42
Iowa Beef calves 12 12,030 0.009% 102 4% 36
Michigan (12:2?ch 0 1549 0% 49 0% 40

 

12

 

Transmission of BVDV Infection

The most efficient means of spreading BVDV is by direct contact with infected
animals, but indirect contact can also occur. Virus has been isolated from nasal swabs,
aerosols, saliva, milk, urine, feces, semen, and uterine fluids. Transmission of BVDV can
be either vertical or horizontal. Vertical transmission from dam to offspring via prenatal
infection is extremely efficient, as discussed above.

The most efficient method of horizontal‘transmission of BVDV is nose-to-nose
contact. Inhalation or ingestion of the virus is the most common mode of infection.
Because PI’s shed large amounts of virus for their entire life, they are considered the
major source of BVDV transmission both within and between herds. Cattle that are
acutely infected are also an important source of transmission of virus. Acutely infected
cattle shed lower levels of virus compared with P1 cattle, and they typically shed the virus
for 2-10 days. Other ruminant species can become infected with BVDV and potentially

serve as a source of transmission. This can include sheep, camelids (llamas and alpacas)

and cervidae (deer and elk).54'58

Indirect transmission of BVDV involves an intermediate vector that transmits the
virus from infected to susceptible animals. Examples of indirect transmission of BVDV
that have been documented include contaminated vaccines or health products,
contaminated needles, biting flies, and other fomites including contaminated equipment,
feed, and people. However, the virus is easily inactivated outside the host, suggesting that
indirect transmission plays a minor role in virus transmission.

Transmission of BVDV has been demonstrated to occur between small ruminants

and cattle, as well as between cervidae and cattle. BVDV has been isolated from pigs and

13

a wide variety of ruminants. Clinical disease has been documented in sheep, alpacas, and
white-tailed deer.54'59 A recent study in Alabama showed that when pregnant deer were
in cohabitation with persistently infected cattle, their offspring were born infected with
BVDV.55 This was the first report of BVDV transmission fiom cattle to white-tailed deer

using a model of natural challenge. Buffalo and wildebeest with no known contact with

cattle have had high BVDV titers detected, suggesting that a wildlife reservoir for BVDV

0

. 6 -62 . . . . . . .
exrsts. The 1mportance of such a reservorr in regards to transmlttrng infectlon to

domestic cattle is unknown.

Economic Importance of BVDV Infection

Bovine viral diarrhea has been reported to be one of the most economically
important diseases in cattle throughout the world.”’45 In addition to losses from fetal

infection (including PI calves and abortions), acute infection can also lead to economic

loss. Losses due to acute forms of BVDV infection include reduced milk production,
reduced conception rate, respiratory disorders and death.45 Through immunosuppression,

BVDV can intensify the effects of BRD pathogens including Mannheimia hemolytica,

. . . . . . 63-67
bovrne herpesv1rus-1, and bovrne resprratory syncytral vrrus.

In the dairy industry, several studies have looked at the economic costs associated
with BVDV infections. Economic losses have been calculated in Canada at $2,421 per 50

head of cattle.68 Estimates of economic losses from infection with highly virulent strains

69,70

range from $40,000 to $100,000 per herd. Other studies have estimated losses at the

population level ranging from $10-40 million per million calvings.36’71

14

To date, only two studies have examined economic effects in the beef industry.
The first was a 10 year study that looked at a farm profitability model.72 This study

calculated an average return to fixed cost of $20. 16 less for farms with at least 1 P1 in the
herd due to reproductive and calf mortality effects. A more recent study evaluated the

economic effects and health and performance of the general cattle population in a starter
feedlot after exposure to P1 cattle.73 This economic analysis of this study revealed that

fatalities accounted for losses of $5.26/animal and performance losses were
$88.26/animal, suggesting that there is a detrimental impact from exposure to BVDV PI

cattle in a feedlot.

Diagnosis of BVDV Infection

Many tests are available to detect BVDV. Test selection is often based on cost
and availability of these. tests. The type of tests available can be broken down into tests
available for detecting individual cattle infected with BVDV and tests available for
detecting infection at the herd level. The identification and removal of BVDV PI animals
from the herd is a key component for controlling of the disease. Several strategies are

available for screening herds for PI’s.

Virus Detection Tests

Individual animal tests are available to diagnose BVDV in the herd. Testing
methods available include virus isolation (V I), immunohistochemistry (IHC), antigen-
capture enzyme-linked immunosorbent assay (ACE), and reverse transcriptase

polymerase chain reaction assays (RT-PCR). Choosing a diagnostic test is based on cost,

15

availability, type of specimen needed, and what type of infection that you are trying to
detect (acute or PI).

Culture and identification of BVDV using VI remains the ‘gold standard’
diagnostic technique. “’75 Several different VI protocols have been developed using three
different cell lines: bovine turbinate, bovine testicle and Madin Darby Bovine Kidney.
The best sample for BVDV isolation in the live animal is the buffy coat (white blood
cells) from a whole blood sample, while lymphoid organs are the best necropsy
specimens. Virus isolation can be affected by colostral irnmunoglobulins, therefore
testing should only be done on calves > 2 months of age.24

Detection of BVDV antigen from samples is much quicker and cheaper than VI.
Two methods are available, antigen capture ELISA’s and immunohistochemistry. IHC
was the first test used to detect BVDV antigen in the skin and is still widely used as a

screening method for identifying PI cattle.76 IHC on formalin-fixed paraffin-embedded

tissues has been shown to be a more sensitive detection method for BVDV antigens in the

skin of cattle when compared with V1.77'79

More recently, ACE has become one of the predominant screening tests due to its
high sensitivity in detection of FPS and ease of use. Serum or skin samples can be used.
Commercially available test kits exist that use monoclonal antibodies to capture viral

antigen E (gp48) and detects antigen-antibody complexes with enzyme-conjugated
antibody by spectrophotometer.80 Previous studies evaluating the use of ACE on skin
samples have shown an high sensitivity and specificity in detecting persistently infected

animals.81’82 Although ACE has been shown to be useful in screening for PI cattle, the

16

procedure lacks the sensitivity needed to detect most acute BVDV infections in cattle.75
In addition, results may be inhibited by passive immunity and is not recommended for

83
young calves.

RT-PCR is a highly sensitive diagnostic test that detects pestivirus RNA.74 RT-
PCR can use skin, serum, or whole blood samples. The high analytical sensitivity of RT-
PCR allows for pooling of specimens.75 Equipment costs to run RT-PCR in a laboratory
setting can be high, but the procedures are readily adapted to pooled sampling strategies,
which reduce test costs to producers. This is especially true when testing for PI cattle,
where studies have detected 1 positive serum sample in pools of 50 and 100 samples and
1 positive animal among 99 negative was detected using pooled supematants from ear

84,85

notch samples. A latter study also calculated a sensitivity of 100% and specificity of

97.5% when using pooled RT-PCR to detect persistently infected cattle.86 The sensitivity

of the RT-PCR assay also increases the likelihood of detecting acute infections.87 RT-

PCR assay of pooled samples is the diagnostic test of choice of the Michigan State
University Diagnostic Center for Population and Animal Health (DCPAH) for BVDV PI

screening.

Herd Tests
Tests are available for detecting infection at the herd level without testing the

whole herd. This can be accomplished by serological evaluation of sentinel animals, bulk

tank RT-PCR, or bulk tank antibody testing.

17

Serologic evaluation of sentinel animals

Sentinel animals are a representative sample of population or herd that serve to
determine the disease status of the herd. Analyzing BVDV neutralizing antibody titers of
a small group of young unvaccinated heifers has been shown to accurately identify herds
infected with BVDV. A high prevalence of seropositive heifers in a herd is indirect
evidence that a PI animal is present in the herd. These animals serve as sentinels for
circulating virus, suggesting the presence of a PI herd mate.

Initial studies in Denmark and Michigan used a hyper geometric probability
fimction for herds with and without PI cattle to calculate the probability of obtaining
seropositive animals. The Danish study looked at the probability of obtaining at least two
out of five seropositive animals 6-18 months of age. The probability was 0.977-1 in 10
herds with P1 cattle and _0-0.048 in nine herds without PI cattle.88 The use of a cutoff of at
least two of five calves being seropositive resulted in a sensitivity of detecting
persistently infected cattle in the herd of 97.7% and a specificity of 95.2%. The Michigan
study determined that the probability of a PI animal being present in a herd was 0.99 if
three out of five animals had BVDV antibody titers 2128. The study further calculated
the probability of a PI animal being in the herd was < 0.01 if three of the five animals had
titers $64.89 The use of a cutoff of at least three of five calves being seropositive resulted
in a sensitivity of detecting persistently infected cattle in the herd of 99.4% and a
specificity of 99%. In both of these studies, all cattle had been tested for both BVDV and
BVDV antibodies prior to the study.

To date, two studies have looked at implementing sentinel animals in herds of

unknown BVDV status to identify herds that contain cattle persistently infected with

18

BVDV. A study of dairy herds looked at 14 herds and used 5 unvaccinated 6-12 month
old heifers as sentinels.9O A herd was classified as likely to contain PI cattle when at least

three out of five heifers had antibody titers 2128. The herd sensitivity for the serological

test was 66% and the specificity was 100%. A separate study looked at 38 beef herds and
collected samples from 30 unvaccinated calves.91 This study determined the optimum

sentinel sample size was 10 animals per herd. A herd was classified as likely to contain
PI cattle when at least three out of ten calves at weaning had antibody titers 21000. The
herd sensitivity for beef herds was 53% and specificity was 80%. The authors concluded
that serological evaluation of a small number of calves could not be used to accurately
predict the presence of PI cattle in a herd. The authors cited potential misclassifications
(vaccination, passive antibodies, comingling with other herds) and environmental factors
(lack of comingling between management groups, low stocking density) as possible
reasons for the lower sensitivity/specificity in their study.

Sentinel testing provides a representation of the entire herd without the cost and
time associated with whole herd testing. It can predict the presence or absence of a

current infection. Unvaccinated calves aged 6-12 months are typically used in sentinel
testing programs.89 Cattle in this age group have typically cleared passively derived

antibodies that could interfere with testing.

One potential negative to sentinel testing is herd misclassification. A false
positive test may occur if the PI animal is removed from the herd shortly before sampling
occurs. A false negative test could occur if a PI animal is extremely young or has been
recently introduced to the herd. In this scenario, the virus may not have time to

disseminate throughout the herd.

19

Bulk tank RT -PCR

Another method of identifying infected herds is the detection of BVDV RNA in
bulk tank milk samples using RT-PCR. RT-PCR is able to identify active BVDV
infections because it detects viral RNA. RT-PCR has been shown to be 14.6 times more .

sensitive than virus isolation and has been proven to be a sensitive and economic method

for the detection of a single PI animal within a group of several hundred cows.92 Drew et

al. was able to detect one PI cow in a herd of 162.93

RT-PCR has an advantage over virus isolation in that the virus does not need to

be replicating for RT-PCR to be positive. It is possible that there will be a high
prevalence of BVDV antibody carriers in a herd where a PI cow is present.92 In this

situation it is possible that these antibodies may inactivate virus in the bulk tank sample,
rendering the virus isolation negative. However, viral RNA will still be detected by RT-
PCR.

There are however, several limitations to bulk tank RT-PCR. One disadvantage is
that it only screens cows whose milk contributed to the bulk tank on the day it was
sampled and is testing is restricted only to the lactating herd. Therefore, bulk tank RT-
PCR cannot report if virus is circulating through the herd. The majority of PI cattle are

young stock, and few survive to become part of the lactating herd.

Bulk tank B VD V antibody testing

In dairy herds, BVDV can be detected by analyzing bulk tank samples for BVDV

94-96

antibodies. This is most commonly done using indirect ELISA. In this instance,

20

results are expressed as optical density absorbance values. The calculated absorbance
value from the indirect ELISA is closely correlated to the prevalence of antibody positive
cows in the lactating herd. In a Swedish study, herds with low absorbance values < 0.20
had a low or zero prevalence of seropositive animals (prevalence range 0-26.5%),

whereas herds with absorbance values > 0.8 had a higher prevalence of seropositive
. 94
animals (87-100%).
Bulk tank ELISA is the foundation for national BVDV control and eradication

programs in many European countries including Sweden, Denmark, Finland, Norway,
and the Netherlands.97 It has also been useful in determining the incidence of BVDV

infection within herds. The advantages of bulk tank ELISA include: an easy to obtain
sample, the ELISA is quick and inexpensive, and the test is closely correlated to the
prevalence of BVDV antibody caries in the lactating herd. The disadvantage of this test is
that it is only a reflection of herd exposure and cannot identify active infection. More
importantly, this method is not practical in North America due to the widespread use of

BVDV vaccines.

Herd Screening Strategies

Several strategies for screening herds have been developed and are used
practically to identify PI cattle and BVDV infected herds. These strategies are
summarized in Table 1.2.

Whole herd screening has been recommended in the past to identify PI cattle in
herds believed to have a problem with BVDV. Because these animals represent less than

1% of the cattle population, the best method to detect a PI animal involves testing an

21

entire herd followed by the testing of every calf born for the nine months proceeding
initial testing and removal of PI animals. Any animal testing positive should be isolated
and retested in three weeks before being classified as persistently infected. Whole herd
testing can be accomplished by detecting virus or viral antigens in infected cattle using
serum, skin, or milk samples.

For producers, Whole herd testing often is not practical, as it is a major
commitment of both time and money. As a result, sub-sampling methods have been
developed for herd surveillance. These include testing newborns and replacement
animals, sentinel animal antibody surveillance, and continued calf surveillance.
Continued calf surveillance involves using one or several of the diagnostics tests
described previously (ACE, IHC, RT-PCR) and evaluates the status of a herd through
sampling of the current calf crop. Surveillance of the calf crop gives the veterinarian and
producer insight into the herd’s BVDV status at that specific point in time and also serves
as a means to test the dam. If a calf is BVDV test negative, it suggests that her cow is
BVDV-free as well. If a calf is BVDV-PI positive, it suggests that the dam is either a P1
or has been exposed to BVDV in the past.

In addition, all replacement animals and their subsequent calves should be tested.
The most common way for BVDV to be introduced into a herd is through the purchase of
new cattle. To prevent these animals from infecting a herd, they should be screened for

BVDV and isolated if possible from the herd until those results are available. In addition,

all newborn calves should be tested from any purchased pregnant cattle.83

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24

Control and Prevention of BVDV

A BVDV control program needs to be both multidimensional and comprehensive
with an overall goal to eliminate BVDV from the herd and to maintain BVDV free status
for years to come. The key components to an effective control program are
biocontainment, biosecurity, and vaccination.

Biocontainment includes the identification and removal of PI cattle fiom the
herd, which is the key component of a BVDV control and prevention program. Once PI
cattle are removed from the herd, circulation of BVDV is essentially stopped.

The goal of biosecurity is to reduce the risk of BVDV being introduced to the
herd by identifying the risks, understanding the importance of each risk, and managing

those risks. Biosecurity can be'broken up into three categories: disease screening

(diagnostic testing), isolation, and sanitation.83

A variety of diagnostic testing strategies are available and were identified
previously (Table1.2). Once BVDV has been detected in a herd and all PI cattle have
been identified and removed (biocontainment), the next step is to prevent the herd from
being reinfected with BVDV. The most common way in which BVDV is introduced into
the herd is through the addition of new cattle. Therefore, strict biosecurity is critical for
maintaining a virus-free herd. Ideally the herd will remain closed, where no outside cattle
are brought into the herd. If not, then all incoming cattle should be tested prior to
introduction to the herd. It is also recommended that replacement cows and heifers are
open, as pregnant cattle can carry PI calves even if they are not infected. If pregnant

animals are purchased, calves should be isolated at birth to determine infection status.

25

Show cattle should also be isolated for 3—4 weeks following their return to the farm, as
these animals are at a high risk of bringing BVDV into the herd.

BVDV typically does not survive outside of its host(s) and is susceptible to
common disinfectants. However the virus has been shown to live in manure and has been
isolated fiom manure up to three weeks at temperatures slightly above fi'eezing (41 F ).98
Therefore, sanitation is important, as precautions should be taken to prevent potential
BVDV contaminated objects (boots, vehicles, and clothing) from entering the premises.

In addition, other ruminants such as sheep, alpacas, and white-tail deer can

54-59

become infected and serve as a source of transmission for BVDV. Therefore,

management strategies to limit or eliminate contact with other ruminants and/or wildlife
should be evaluated.
Vaccination plays an important role in the control of BVDV. Although no vaccine

is 100% efficacious, vaccination is an option for controlling the spread of virus and
reducing the risk of infection in herds.83 Vaccination also has a role in preventing acute

infections that can result in severe disease, as well as reducing the risk of fetal infection.
Use of the BVDV vaccine is a sound management practice to reduce the risks associated
with BVDV infections. Both killed and modified-live virus (MLV) BVDV vaccines are

available. In general, MLV vaccines are believed to be more effective. Vaccine
attributes are summarized in Table 1.3.83 Vaccination protocols are summarized in Table

1.4, but all vaccines should be used according to labeled directions.

26

Table 1.3 BVDV vaccine attributes

 

Vaccine Type

Advantages

Disadvantages

 

Killed

0 Safe in all cattle
0 Individual doses of
vaccine can be removed

from bottles over time

o Shorter duration of

immunity

 

 

Modified-live (MLV)

 

0 Rapid response

0 Can induce immunity
with a single dose

0 Broader protection

0 Longer duration of

immunity

 

0 Can cause abortion
0 MLV vaccines must
be used within 4 hours

after reconstitution

 

27

 

Table 1.4 BVDV vaccination strategies

 

Vaccination of calves to prevent subsequent disease

 

Most Reliable

Least Reliable

 

 

Vaccination after four months of age with two doses of modified
live vaccine four weeks apart on the farm of origin prior to
weaning, transport, and commingling.

Vaccination after four months of age with a single dose of
modified live vaccine on the farm of origin prior to weaning,
transport, and commingling.

Vaccination after four months of age with two doses of killed
vaccine four weeks apart on the farm of origin prior to weaning,
transport, and commingling.

Vaccination prior to four months of age with a single dose of
modified live vaccine to healthy calves that nursed adequate
colostrums

Vaccination after four months of age with a single dose of killed
vaccine on the farm of origin prior to weaning, transport, and
commingling.

Vaccination prior to four months of age with a single dose of
modified live or killed vaccine to healthy calves that nursed
adequate colostrum.

 

Vaccination of heifers and cows to prevent reproductive losses

 

 

Most Reliable

 

1

 

Vaccination of heifers with two doses of modified live vaccine at
least 30 days before initial breeding, and annual revaccination
with a single dose of modified live vaccine prior to breeding or at
branding or weaning.

Vaccination of heifers with two doses of modified live vaccine at
least 30 days before initial breeding, and annual revaccination
with a single dose of killed vaccine at branding or weaning.

 

28

 

Table 1.4 - continued

 

Vaccination of heifers with one dose of modified live vaccine at
least 30 days before initial breeding, and annual revaccination
with a single dose of modified live vaccine prior to breeding or at
branding or weaning.

 

Vaccination of heifers with one dose of modified live vaccine at
least 30 days before initial breeding, and annual revaccination
With a single dose of killed vaccine at branding or weaning.

 

Vaccination of heifers with two doses of killed vaccine at least 30
days before initial breeding, and annual revaccination with a
single dose of killed vaccine prior to breeding or at branding or
weaning.

 

Least Reliable

 

 

 

Vaccination of heifers with two doses of killed vaccine at least 30
days before initial breeding, and annual revaccination with a
single dose of killed vaccine prior to breeding or at branding or
weaning.

Vaccination of heifers with one or two doses of modified live
vaccine at least 30 days before initial breeding, without annual
revaccination

Vaccination of heifers and cows each year prior to breeding with a
single dose of killed virus.

 

Eradication Programs

Eradication programs have been in place in Europe since the 1990’s.99 Programs

have been put in place in Denmark, Sweden, Norway, Finland, Austria, Germany, and

Switzerlandloo'los

The success of these programs led to the establishment of an

eradication program in the United States. In Michigan, the Upper Peninsula BVDV

eradication program is designed to eradicate the virus for a particular region in Michigan.

29

 

One of the goals of the eradication program was to develop effective and economical
herd testing strategies such as sentinel antibody testing.
In European BVDV eradication programs, bulk tank antibody testing is the main

focal point of most eradication programs. In addition, spot testing is employed in beef
herds as well as in follow-up testing of dairy herds.100 Spot testing involves randomly

selecting young animals for antibody testing to predict the presence or absence of
infection in a herd. This is based on the high probability of seropositivity in groups of
animals where PI animals are present.36 In Europe, serological testing of random calves
using ELISA is part of eradication programs in Denmark, Sweden, Norway, and Austria.
The eradication programs in these countries do not involve the use of vaccines and calves
selected for testing ranged from 6-12 months of age depending on that nation’s

program.38’99’100 In addition, eradication programs in Sweden and Austria take into

account herd size when selecting sentinel animals. In Sweden, individual serum samples
are collected from 5, 8, or 10 young stock at 12 months of age.100 The number of animals
chosen depends on herd size. In Austria, 15% or at least 5 animals from the young stock
are tested serologically twice yearly to test for herd BVDV status.103 In addition, sentinel
testing of calves is seen in Germany where an on-going eradication program includes
97,100,109

vaccination.

The success of these national programs suggest that serological testing of young
stock is a valid and useful screening method that can be applied to in an eradication

program here in the United States.

30

Conclusion

BVDV is one of the most economically damaging diseases of cattle. Its complex
nature and various clinical manifestations have made control and prevention of BVDV a
challenging undertaking. Implementation of proper control measures such as biosecurity
and a proper vaccination may reduce the incidence and/or severity of BVDV in a herd.
The implementation of a proper screening program is also important for controlling
BVDV, although it can prove to be costly to the owner. The potential for less expensive
follow-up screening programs is something that should be further pursued in these
changing economic times. As eradication projects begin in the United States, less
expensive, efficacious follow-up screening programs are something that should be further
pursued. One such screening program is the use of sentinel testing, as sentinel animals are
a representative sample of a herd that serves to determine the disease status of the herd.
The success of national screening programs in Europe suggest that serological testing of
young stock is a valid and useful screening method that could be employed in the United

States in the form of sentinel animal antibody testing.

31

10.

11.

12.

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infected with BVDV. J Vet Diagn Invest 1995;7z327-332.

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4O

CHAPTER 2
SEROLOGICAL EVALUATION OF SENTINEL CALVES IN A BVDV
ERADICATION PROGRAM

Abstract

Objectives. To evaluate serology as a tool to detect herd infection with BVDV as part of
. an eradication program.

Sample Population. FOrty-seven cattle management groups from 36 herds in a regional
BVDV Eradication Program.

Procedure. Serum samples were obtained from five non-vaccinated sentinel calves 26
months old in each management group and VN antibody titers against BVDV genotypes
1 and 2 were determined. A herd was considered positive if two or more sentinel calves
had VN antibody titers 2128 to either genotype. Results were compared to individual
animal testing of all available calves by RT-PCR on skin samples.

Results. In one management group, 3 sentinel calves had VN antibody titers 2128.
Three ear notch samples from that herd were positive for BVDV on RT-PCR assay. All
other management groups were negative for BVDV. In this study, the herd sensitivity of
sentinel serology was 100% (95% CI, 0.05 to 1.0) and herd specificity was 100% (95%
CI, 0.90 to 1.0). The K value for agreement between sentinel serology and RT-PCR was
1.0 (95% CI 1.0 to 1.0).

Conclusion. Sentinel animal serology can be utilized in a BVDV eradication program to
provide an accurate and efficient evaluation of herd status. Although the sensitivity and
specificity of serologic surveillance were high in this study, the approach used here may

not be suitable for other. geographical locations and cattle management systems.

41

Introduction

Control of bovine viral diarrhea virus (BVDV) has been a worldwide challenge
for decades. This virus causes substantial economic loss to the cattle industry, ranging
from $10-40 million per" million calvings.l'3 Various clinical manifestations can result
from infection with BVDV, ranging from subclinical infection to immunosuppression

and respiratory disease to abortion and infertility.4'7 The most well known source for

transmission of BVDV are persistently infected (PI) cattle.l’2’8 Persistent infection
occurs when calves are exposed to BVDV in utero before the fetal immune system is
developed, thus allowing the developing fetus to become immunotolerant to the virus.5

PI cattle continuously shed large amounts of virus into the environment and are an
important source of virus transmission within and between herds.

Because of the clinical and economical importance of BVDV, many European
countries have implemented BVDV eradication programs including Denmark, Sweden,

Norway, Finland, Austria, Germany and Switzerland?’17 In Michigan, the Upper

Peninsula BVDV Eradication Program was launched in 2008 and with the goal of
eradicating BVDV fi'om a region within the US. This integrated program included
education, identifying and eliminating PI’s, vaccination and biosecurity.

A key step in controlling and eradicating BVDV from a herd is the ability to
identify and remove all PI cattle. Multiple diagnostic tests have been developed to
identify PI cattle including virus isolation (VI), antigen capture ELISA (ACE),

immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-

42

PCR). Unfortunately, whole herd testing using any of these tests can be a major
investment of both time and money for producers. Therefore developing and
implementing alternative herd screening tests would be beneficial for identification of
infection with BVDV at the herd level before embarking on individual animal testing.
Serological evaluation of small groups of young non-vaccinated calves using

virus neutralization (VN) or ELISA has been proposed as a method for identifying herds

with PIcattle.18'22 The premise is that a high prevalence of seropositive calves in a herd

. . . . . . 20 . . .
rs indirect evrdence that a PI animal 18 present. The calves serve as sentinels for vrrus

being shed by a PI herd mate. Calves selected as sentinels should be non-vaccinatedand
26 months old because colostral antibodies may be present in the serum of calves < 6
months old.

Initial studies in Denmark and Michigan used a hyper geometric probability

firnction for herds with and without PI cattle to calculate the probability of detecting

18’” The Danish study evaluated sera using indirect ELISA and

seropositive animals.
assessed the probability of detecting PI animals when at least two out of five sentinel
animals were seropositive at 6-18 months of age.18 Using that seropositive rate, a
predicted sensitivity of 97.7% was proposed for detecting herds containing at least one PI
animal. In addition the predicted specificity was 95.2%. The Michigan study evaluated
sera using VN titers and determined that the probability of a PI animal being present in a

herd was 0.99 if three out of five animals had BVDV antibody titers 2128. The study

further calculated the probability of a PI animal being in the herd was < 0.01 if three of

the five animals had titers $64.19 In both studies, all cattle had been tested for both

BVDV and BVDV antibodies prior to the study.

43

Use of sentinel animals in herds of unknown BVDV status to detect presence of
cattle persistently infected with BVDV also has been reported. In one study, dairy herds

were considered likely to contain PI cattle when at least three out of five heifers, 6 to 12
months old had VN antibody titers 2128.21 The sensitivity of using VN titers from
sentinel calves to detect BVDV was 66% and the specificity was 100%. Another study
done in beef cattle evaluated use of 10 non-vaccinated calves per herd.22 A herd was

classified as likely to contain PI cattle when at least three out of ten calves at weaning
had antibody titers 21000. Employing this measure, the sensitivity for use of VN titers in
sentinel beef calves to detect the presence of a PI animal in a herd was 53% and
specificity was 80%.

In Europe, serological testing of random calves using ELISA is part of eradication

9-12,l4-18,23

programs in Denmark, Sweden, Norway, Finland and Austria. The eradication

programs in these countries do not involve the use of vaccines and calves selected for
testing range from 6-12 months of age depending on that nation’s program. In addition,
sentinel testing of calves is used in Germany where an on-going eradication program

. . . 9,24-26
includes vaccrnatron.

The objective of the study reported here was to evaluate the application of
sentinel serology as a herd monitoring tool to detect herd infection with BVDV as part of

an eradication program in the Upper Peninsula of Michigan.

44

Materials and Methods

Selection of Herds - Herds (n = 33) enrolled in the Michigan Upper Peninsula BVDV
Eradication Program that had completed whole herd BVDV testing in 2008 were asked to
voluntarily participate and agree to a sentinel testing scheme as well as a retest of the
whole herd in 2009. Twenty-nine herds had tested negative for BVDV in 2008 and 4
herds had at least one calf persistently infected with BVDV diagnosed in 2008. Thirty-
one of the herds were beef, one was dairy, and one was mixed dairy and beef. An
additional 3 herds (beef) volunteered to participate that were having whole herd tests for
the first time in 2009. Some (11 = 6) of the 36 herds had more than 1 management group,
which was defined as groups of animals on the farm that were managed separately from

other animals or groups.

Sample Collection - Skin samples were collected fiom the ear of calves shortly after
birth or at weaning by uSe of 5/16 ear notcher and then placed in individually labeled
bags. These samples were placed on ice or in a refrigerator for storage before being
submitted within one week of collection to the Michigan State University Diagnostic
Center for Population and Animal Health (DCPAH). The ear notch samples were
obtained over the course of 2009. Serum samples were obtained from five non-vaccinated
calves 26 months old in each herd or, in the case of herds with multiple management
groups, each management group within a herd. Collection of serum was done from late
summer through late fall of 2009. Samples were placed on ice or in a refiigerator prior to

being submitted to DCPAH within one week of collection.

45

Herd Infection Definition - A herd was considered to be infected with BVDV based on
the following criteria: .

1. If 2 of 5 calves in any management group had VN antibody titers 2128 for either
genotype 1 or 2 BVDV. Conversely, if 4 of 5 calves in all management groups
had VN titers _<_ 64 to both genotypes 1 and 2 BVDV, the herd was considered
negative for BVDV.

2. A herd was considered positive if any animal in any management group was

found to be positive for BVDV by RT-PCR assay on skin samples.

RT-PCR - Skin samples were analyzed at DCPAH using their standard pooled (n 510
ear notches per pool) RT-PCR assay, a hydrolysis probe-based real-time, RT-PCR assay
with an internal RNA control to monitor inhibition of the reaction was used to detect
RNA from BVDV. The BVDV-specific primers and probe were designed from conserved

sequences within the 5’ UTR. The assay amplifies both BVDV genotypes 1 and 2 based

upon testing of a validation panel provided by Dr. J. Ridpath (NADC, Ames, IA).27 The

detection limit of the assay is 0.8 TCIDso.28 If a pool of ear notches was positive,

individual samples contained in the pool were confirmed by fluorescent antibody testing
of fresh frozen sections of tissue. Any animal positive on initial testing was retested a

minimum of 14 days later to confirm persistence of the virus.

Virus Neutralization - Serum samples were tested for VN antibodies against both
genotypes l and 2 BVDV using standard microtitration assay procedures. Cytopathic

Singer strain was used as the genotype 1 BVDV reference strain and cytopathic 125C

46

strain as the genotype 2 BVDV reference strain. The VN test was conducted using bovine

29-31

turbinate cells that were free of adventitious BVDV. The fetal bovine serum

supplement for cell culture medium were also free of live adventitious BVDV, RNA from
BVDV, and antibody against BVDV. Serial 2-fold dilutions ranging from 1:4 to 1:4,096
were made for each sample of serum. Antibody titer was considered to be the highest
serum dilution at which the cytopathic effect of the BVDV reference strain was

completely inhibited.

Data Analysis - Information collected included number of management groups, herd
history, and ear notch results. Data was described using descriptive statistics. The
sensitivity and specificity of the sentinel calf testing strategy was determined using
results of the RT-PCR assay on car notches as the gold standard. Correlation between the
RT-PCR assay and sentinel animal serology as herd based tests was calculated using the

K test statistic.
Results

Herds - Thirty-six herds consisting of forty-seven management groups were included in
the study. Ear notch samples were collected from 2,206 cattle during 2009. A total of 395
serum samples were collected in the summer and fall of 2009. Serum samples were
collected from calves that were not vaccinated and at least 6 months old at the time of
sampling. Description data and results from the herds tested are summarized in Table 2.1.

Ear notches from three calves from one management group were positive for BVDV.

47

Those calves were confirmed to be persistently infected with BVDV on follow-up
testing. The same management group was also positive for BVDV based on VN titers of
_>_ 128 from 3 of 5 sentinel calves for BVDV genotype 1 and 2. The other forty-six
management groups tested did not contain PI cattle based on either RT-PCR or VN

results.

Analysis of Serologic Data - VN results from non-vaccinated sentinel calves from each
management group were compared with the results for herd testing using RT-PCR. The
herd sensitivity for VN testing was 100% (95% CI, 0.05 to 1.0) and herd specificity was
100% (95% CI, 0.90 to 1.0). The K value for agreement between the two tests was 1.0
(95% CI 1.0 to 1.0). To determine the influence of changing the VN cutoff value or the
number of calves above or below the VN cutoff value on the accuracy of this strategy,
results of serologic testing from each management group were compared with various
cutoff values for number of positive calves and minimum antibody concentration. As the
number of positive calves and minimum antibody concentration were lowered, the
diagnostic specificity of the test decreased (Table 2.2). The sensitivity remained

unchanged throughout.
Discussion

The results from this study support and extend earlier studies which found sentinel
serologic testing useful for screening herds for infection with BVDV.19’21’22 In the

current study, serological evaluation of 5 non-vaccinated calves per management

48

group was an accurate herd screening method for predicting the presence of PI cattle in a
herd. There was 100% correlation between detection of PI cattle in a herd using RT-PCR
on ear notches and the predicted presence of PI cattle in a herd using sentinel serology.

In this study, when a management group had a positive result fi'om sentinel
serology using pre established cutoff criteria, a P1 was present in the herd and a negative
result correlated with no PI’s in the management groups. However, since only one

management group was positive in the study, the sensitivity cannot be accurately

assessed. Previous studies have reported lower sensitivities than that reported here.” ’22

Pillars and Grooms found a sensitivity of 66% in dairy herds when using a diagnostic

criteria of 3 of 5 calves with VN antibody titers 2128.21 If they had changed their

diagnostic criteria to 2 of 5 calves, the sensitivity would have improved to 83%.32 In that

study the diagnostic cutoff criteria was selected based on cost. It was determined that the
cost of extensive testing associated with a false positive result was of more concern than
the cost of a false negative result. Changing the diagnostic criteria used to define a
positive herd would alter the sensitivity and specificity of the test. Sensitivity can be
improved by decreasing the VN antibody titer that defines an individual animal as
positive. Sensitivity can also be improved by decreasing the number of sentinel calves
that have antibody titers above a designated cutoff. Since the goal of surveillance in an
eradication program is to identify any and all potentially positive herds, a premium is
placed on sensitivity. In this study, as well as in the study done by Pillars and Grooms,
decreasing the number of calves which needed to have a minimum titer of 2128 from 3

to 2 increased sensitivity but did not affect specificity.

49

Reasons for lowered specificity of sentinel serology discussed in previous studies
include vaccination of sentinel calves, persistence of passively acquired antibodies, and
acute infections introduced by breakdowns in biosecurity. To address these potential
problems, the target population for the study reported here were unvaccinated calves that
were 6 to 12 months of age. By testing calves older than 6 months of age, the risk of

detecting passively acquired antibodies is decreased significantly as these antibodies are
usually undetectable by 6 months of age.18 In addition, all herds involved in this study

were implementing biosecurity measures to reduce the risk of herd infection. This
included reducing contact with neighboring cattle farms and screening new cattle for
BVDV. These practices help to reduce the risk of herd misclassification

Herd size, multiple management groups and low stocking densities have been
cited in previous studies as reasons for lower sensitivity of sentinel serology as a herd
based assay for BVDV. Previous studies conducted in dairy herds showed that herd size
did not impact the results of serologic testing and suggested that evaluation of five non-
vaccinated calves would be effective for herds of varying size as long as the herd was

l8,l9,21,23

managed as one unit with commingling of cattle of various ages. Since most of

the herds in this study (35 of 36 herds) were beef, serologic testing of any and all
management groups was done to increase the sensitivity of the test. In addition low
stocking density should have been less of a factor, as herds are more contained in the
Upper Peninsula of Michigan.

In European BVDV eradication programs, serological testing is part of many

eradication programs. This is based on the high probability of seropositivity in groups of

animals where PI animals are present.1 In Europe, serological testing of random calves

50

using ELISA is part of eradication programs in Denmark, Sweden, Norway, and Austria.
The eradication programs in these countries do not involve the use of vaccines and calves

selected for testing ranged from 6-12 months of age depending on that nation’s

program.8’9’33 In addition, eradication programs in Sweden and Austria take into account

herd size when selecting sentinel animals. In Sweden, individual serum samples are
collected from 5, 8, or 10 young stock at 12 months of age.9 The number of animals
chosen depends on herd size. In Austria, 15% or at least 5 animals fiom the young stock
are tested serologically twice yearly to test for herd BVDV status.12 The success of these
national programs suggest that serological testing of young stock is a valid and useful
screening method that can be applied to in an eradication program here in the United
States.

Results from this study provide supportive evidence that serological evaluation of
non-vaccinated calves can be utilized as a screening tool for herd infection with BVDV
and could be employed as an accurate and economically viable surveillance tool to
evaluate herds in a BVDV eradication program. Although the strategy was highly
sensitive and specific in, the herds assayed for this study, application to herds in different

parts of the country and using different management strategies should be evaluated.

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1].

12.

13.

References

. Houe H. Epidemiology of bovine viral diarrhea virus. Vet Clin North Am Food

Anim Pract 1995;11:521-547.

Houe H. Epidemiological features and economical importance of bovine virus
diarrhoea virus (BVDV) infections. Vet Microbiol 1999;64:89-107.

Bennett RM, Christiansen K, Clifton-Hadley RS. Direct costs of endemic diseases
of farm animals in Great Britain. Vet Rec 1999;145:376-377.

Baker J C. The clinical manifestations of bovine viral diarrhea infection. Vet Clin
North Am Food Anim Pract 1995;11:425-445.

Chase CC, Ehnowalid G, Yousif AA. The immune response to bovine viral
diarrhea virus: a constantly changing picture. Vet Clin North Am Food Anim Pract
2004;20:95-114.

Grooms DL. Reproductive consequences of infection with bovine viral diarrhea
virus. Vet Clin North Am Food Anim Pract 2004;20:5-19.

Loneragan GH, Thomson DU, Montgomery DL, et al. Prevalence, outcome, and
health consequences associated with persistent infection with bovine viral
diarrhea virus in feedlot cattle. J Am Vet Med Assoc 2005;226:595-601.

Lindberg A, Houe H. Characteristics in the epidemiology of bovine viral diarrhea
virus (BVDV) of relevance to control. Prev Vet Med 2005;72:55-73.

Houe H, Lindberg A, Moennig V. Test strategies in bovine viral diarrhea virus
control and eradication campaigns in Europe. J Vet Diagn Invest 2006;18:427-
436.

Hult L, Lindberg A. Experiences from BVDV control in Sweden. Prev Vet Med
2005;72:143-148.

Lindberg A. The Nordic bovine virus dirrhoea eradication programmes - their
success and future. Cattle Pract 2004;12:3-5.

Obritzhauser W, Klemens F, Josef K. BVDV infection risk in the course of the
voluntary BVDV eradication program in Styria/Austria. Prev Vet Med
2005;72:127-132.

Presi P, Heim D. BVD eradication in Switzerland--a new approach. Vet Microbiol
2010;142:137-142

SS

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Rikula U, Nuotio L, Aaltonen T, et a1. Bovine viral diarrhoea virus control in
Finland 1998-2004. Prev Vet Med 2005;72:139-142.

Rossmanith W, J anacek R, Wilhelm E. Control of BVDV-infection on common
grassland--the key for successful BVDV-eradication in Lower Austria. Prev Vet
Med 2005;72:133-137.

Rossmanith W, Deinhofer M, J anacek R, et al. Voluntary and compulsory
eradication of bovine viral diarrhoea virus in Lower Austria. Vet Microbiol
2010;142:143-149.

Valle PS, Skjerve E, Martin SW, et al. Ten years of bovine virus diarrhoea virus
(BVDV) control in Norway: a cost-benefit analysis. Prev Vet Med 2005;72:189-
207.

Houe H. Serological analysis of a small herd sample to predict presence or
absence of animals persistently infected with bovine viral diarrhoea virus
(BVDV) in dairy herds. Res Vet Sci 1992;53:320-323.

Houe H, Baker J C, Maes RK, et a1. Application of antibody titers against bovine
viral diarrhea virus (BVDV) as a measure to detect herds with cattle persistently
infected with BVDV. J Vet Diagn Invest 1995;7:327-332.

Houe H, Baker J C, Maes RK, et al. Prevalence of cattle persistently infected with
bovine viral diarrhea virus in 20 dairy herds in two counties in central Michigan
and comparison of prevalence of antibody-positive cattle among herds with
different infection and vaccination status. J Vet Diagn Invest 1995;7z321-326.

Pillars RB, Grooms DL. Serologic evaluation of five unvaccinated heifers to
detect herds that have cattle persistently infected with bovine viral diarrhea virus.
Am J Vet Res 2002;63:499-505.

Waldner CL, Campbell JR. Use of serologic evaluation for antibodies against
bovine viral diarrhea virus for detection of persistently infected calves in beef
herds. Am J Vet Res 2005;66:825-834.

Houe H. Bovine-virus diarrhea virus: detection of Danish dairy herds with
persistently infected animals by means of a screening test of 10 young stock. Prev
Vet Med 1994;19:241-248.

Moennig V, Houe H, Lindberg A. BVD control in Europe: current status and
perspectives. Anim Health Res Rev 2005;6263-74.

Lindberg A, Brownlie J, Gunn GJ, et al. The control of bovine viral diarrhoea
virus in Europe: today and in the future. Rev Sci Tech 2006;25:961-979.

Sandvik T. Progress of control and prevention programs for bovine viral diarrhea
virus in Europe. Vet Clin North Am Food Anim Pract 2004;20:151-169.

56

27.

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Ridpath JF, Bolin SR. Differentiation of types 1a, 1b and 2 bovine viral diarrhoea
virus (BVDV) by PCR. Mol Cell Probes 1998;12:101-106.

Wise A.G, Wu P., Benson C., et al. A taqman-based real-time RT-PCR with
internal control for the detection of BVD virus in ear notch samples. in
Proceedings. 49th Annu Meet Am Assoc Vet Lab Diag 2006;35.

Bolin SR, Matthews PJ, Ridpath JF. Methods for detection and frequency of
contamination of fetal calf serum with bovine viral diarrhea virus and antibodies
against bovine viral diarrhea virus. J Vet Diagn Invest 1991 ;3: 199-203.

Bolin SR, Ridpath JF, Black J, et al. Survey of cell lines in the American Type
Culture Collection for bovine viral diarrhea virus. J Virol Methods 1994;48:211-
221.

Bolin SR, Ridpath JF. Prevalence of bovine viral diarrhea virus genotypes and
antibody against those viral genotypes in fetal bovine serum. J Vet Diagn Invest
1998;10:135-139.

Pillars RB. Serological evaluation of five unvaccinated heifers to detect herds
with cattle persistently infected with bovine viral diarrhea virus. MS Thesis.
Michigan State University. 2001.

Lindberg AL, Alenius S. Principles for eradication of bovine viral diarrhoea virus
(BVDV) infections in cattle populations. Vet Microbiol. 1999;64:197-222.

57

CHAPTER 3
IMMUNIZATION OF CALVES WITH A MODIFIED-LIVE BOVINE VIRAL
DIARRHEA VIRUS VACCINE: EVALUATION OF BOVINE VIRAL
DIARRHEA VIRUS IN CLINICAL SAMPLES POST VACCCINATION

Abstract

Objectives. To determine if and for how long vaccine virus can be detected using RT-
PCR in skin samples following vaccination with a commercially available modified-live
BVDV vaccine

Sample Population. Two different experiments were conducted in this project.
Experiment 1 involved 12 BVDV seropositive steer calves, while experiment 2 involved
7 BVDV seronegative heifers.

Procedure. Skin samples were collected in both experiments at days 0 and 3-18 for virus
detection using both individual and pooled RT-PCR. In parallel, blood and nasal swabs
were collected for virus isolation.

Results. All cattle, regardless of their serological status, were negative for BVDV using
individual and pooled RT-PCR on skin samples. Virus was detected by virus isolation in
5 of 7 of the experiment 2 heifers. In addition, all heifers in experiment 2 seroconverted
to BVDV.

Conclusion. These findings provide evidence that it is highly unlikely that vaccine virus
can be detected in skin by either individual or pooled RT-PCR following vaccination

with a commercially available BVDV vaccine.

58

Introduction

Bovine viral diarrhea virus (BVDV) is an economically important pathogen

affecting cattle worldwide. Economic losses associated with BVDV infection have been
estimated in the range of $10-40 million per million calvings.l A recent study of the
economic effects on reproduction and performance in commercial cow herds showed that
over a ten year period, calculated an average return to fixed cost of $20.16 less for farms
with at least 1 P1 in the herd due to reproductive and calf mortality effects.2 Another
recent study has shown that in feedlots, cattle exposed to persistently infected (PI)
animals cost the producer an average of $88.26 per animal in performance losses.3

Cattle persistently infected with BVDV are the major reservoir for transmission of
virus within and between farms. It is estimated that 4% of cow-calf herds and 15% of
dairy herds have persistently infected (PI) animals in them.4 Identification and removal of

BVDV PI animals from the herd is a key component for controlling virus transmission.

Many different virus detection methods have been used to detect PI’s. Currently, the most
frequently used methods rely on testing of skin for the presence of BVDV.S Methods to

detect BVDV in skin include immunohistochemistry (II-1C), antigen-capture enzyme-
linked immunosorbent assay (ACE), and reverse transcriptase polymerase chain reaction
(RT-PCR) assays. Irnmunohistochemistry was the first test used to detect BVDV antigen
in the skin. More recently, ACE and RT-PCR have become the preferred screening tests
due to their lower cost, ease of use, adaptation for high volume testing, and analytical

sensitivity. RT-PCR has potential for detection of small concentrations of virus, thereby

59

allowing pooled sampling strategies, which reduce test costs to producers. The high
analytical sensitivity of the RT-PCR assay also increases the likelihood of detecting
transient infections.

Vaccination programs are a major component of control and prevention strategies
for BVDV. While modified-live vaccines (MLV) have become the vaccine of choice for
immunization against BVDV, questions have arisen as to whether use of MLV’s could
lead to false positive results on virus detection assays such as those described above.
Several studies have explored the temporal persistence of vaccine virus in various clinical
specimens following vaccination with a modified-live virus. BVDV vaccine virus has
been shown to remain in the ovaries for up to 12 days after vaccination and a recent study
has shown that it can be shed in semen up to 10 days after vaccination.6’7 Previous
studies have shown that when an animal is acutely infected with BVDV, the likelihood of

detection of viral antigen in a skin biopsy by IHC or ACE, is very low.8’9 Other studies

have used RT-PCR to test serum and nasal swab samples from calves vaccinated with a

modified-live virus vaccine for BVDV. In one study, serum samples from 78% of calves
vaccinated were positive for BVDV between 3 and 10 days post-vaccination.IO However

that same study noted that none of the calves sampled had positive results for BVDV
using RT-PCR on nasal swabs. While we know from previous studies that BVDV

vaccine virus can be detected in blood and certain tissues, to date it has not been found in
skin using IHC or ACE.9’ll It is important to determine if vaccine virus can be detected in

the skin because a false positive result for BVDV can lead to more follow-up testing and

more costs to the producer. The objective of this study was to determine if and for how

60

long vaccine virus can be detected using RT-PCR in skin samples following vaccination

with a commercially available MLV BVDV vaccine.
Materials and Methods

Two different experiments were conducted in this project using calves known to
be fi'ee of persistent infection with BVDV.

0 Experiment 1: Twelve BVDV seropositive steer calves (four to five months of
age), were administered a commercially available MLV cytopathic BVDV
vaccinea

0 Experiment 2: Seven BVDV seronegative heifers (eleven months to two and a

half years of age), were administered a commercially available MLV cytopathic
BVDV vaccine":l

Each group was housed in an isolated pen at least 150 yards from other cattle on the

premises.

Clinical Samples

Skin, blood, and nasal swabs were collected from each animal on day 0 (prior to
vaccination) and then on days 3 through 14, 16, and 18 post vaccination. Skin samples
were collected from the ear using a 5/ 16 inch ear notcher and then placed in individually
labeled bags. Serum samples were collected from all cattle by jugular venipuncture into a
serum separator tube. Nasal swabs were collected and placed into tubes containing 1 ml

of Dullbecco’s modified Eagle medium (DMEM). In experiment 2, whole blood samples

61

were obtained using EDTA as an anticoagulant. Samples were placed on ice and
transported to the laboratory where skin samples were halved and placed in separately
labeled bags. Skin, serum and nasal swabs specimens were stored briefly at -20°C until
testing. White blood cells were isolated from whole blood samples collected in
experiment 2 as follows: after collection, samples were stored at room temperature for
one hour and then centrifirged at 1200 rcf for 15 minutes. Following centrifugation, the
buffy coat was collected fiom each sample using individual sterile 1 ml pipettes and
placed into labeled 15 ml polypropylene centrifuge tube. The buffy coat with
contaminating red blood cells was suspended 3 ml of sterile cell culture grade water for
30 to 45 seconds to lyse the red blood cells, and then 1.5 ml of 2X concentrated
physiologic phosphate buffered saline (PBS) was added to the tube. Cells were vortexed
and pelleted by centrifugation at 270 ref for 12 min. A second exposure with 1.5 ml of
sterile water, followed by addition of 0.75 ml of 2X concentrated physiologic PBS was
done. The cells were pelleted at 270 rcf for 12 min, suspended in 0.5 ml of Bovamick’s
solution and frozen at -70°C.

Immediately following vaccination, biological cloning of the viruses contained in
the vaccine was performed. This was done in order to compare any strains of BVDV

recovered in the samples taken with the strains contained within the vaccine.

Polymerase Chain Reaction Assays - All skin samples submitted to the Diagnostic
Center for Population and Animal Health (DCPAH) at Michigan State University. Half
of each ear notch was processed for routine diagnostic testing, which was done using

pools of homogenate from 10 ear notches for RNA extraction. The RNA was then tested

62

by a hydrolysis probe-based real-time RT-PCR that targeted the 5 ' untranslated (UT R)

region of the BVDV genome. An internal RNA control was used to monitor inhibition of
the RT-PCR reaction. The detection limit of the assay is 0.8 TCIDSO.12 Laboratory

protocol requires all individual samples in a positive pool of ear notches be retested using
fluorescent antibody staining of fresh frozen sections of tissue. All individual samples in
a negative pool of ear notches were retested using a gel based RT-PCR targeting the same
5 ' UTR region of the viral genome. A positive ear notch would be confirmed using

fluorescent antibody staining of fresh frozen sections of tissue.

Virus Isolation - Virus isolation was performed on samples of serum, nasal swabs
immersed in 1 ml of transport medium, and white blood cells harvested from whole blood
by centrifirgation followed by selective lysis of red blood cells. Approximately 200uL of
each sample was inoculated onto bovine turbinate (BT) cells in 24-well flat-bottom cell
culture plates. Nasal swab transport medium was passed through a 0.45pm syringe filter
prior to inoculation. The BT cells were free of adventitious BVDV, as determined by RT-
PCR assay. The growth medium for the BT cells was free of adventitious BVDV and
antibody against BVDV as determined by virus isolation, RT-PCR assay, and virus
neutralization assay against type 1 and type 2 BVDV. The cell monolayers were observed
for cytopathic effect (CPE) and any potential BVDV isolations were subpassaged after 5

to 8 days to fresh monolayers of BT cells. After 2 to 5 days, dependent on appearance of
CPE, RNA was extracted from infected cells using TRIzol b as recommended by the

manufacturer. Presence of BVDV was confirmed, and the genotype of the isolate

predicted, using RT-PCR assays that targeted the 3 'end of genomic region encoding viral

63

protein NSSB and most of the 3 'UT R region of the viral genome 13’” The RNA from
select viral isolates was amplified to obtain nucleic acid sequence that included about 100
bases of the 3 ' end of the 5 ' UTR region and all of the genomic region encoding Np'r0 and

Capsid viral proteins. To obtain vaccine virus, genotype 1 and genotype 2 BVDV were
biologically cloned from reconstituted vaccine. This was done by diluting the vaccine
100 fold in cell culture medium, and then serial 4 fold dilutions were made from the
diluted vaccine. The serial dilutions were inoculated onto BT cells that had been seeded
into 96 well microtitration plates. Culture fluid was harvested from wells showing CPE
typical of BVDV and used to inoculate 24 well plates. After appearance of CPE, RNA

was extracted from infected cells and amplified for nucleic acid sequencing.

Genetic Analysis - Thesamples of amplified nucleic acid were submitted to the

Michigan State University Research Technology Support Facility for DNA sequencing.
Sequences were aligned and trimmed using software described previously.15

Phylogenetic analyses were conducted by use of integrated software as previously

described. 16

Virus Neutralization - Serum samples were tested for viral neutralizing (VN) antibodies
against genotypes 1a, 1b and 2a BVDV using standard microtitration assay procedures.
The cytopathic Singer strain was used as the genotype 1a BVDV reference strain,
cytopathic strain TGAC as the genotype 1b reference strain, and cytopathic 125C strain

as the genotype 2a BVDV reference strain. The VN test was conducted using bovine

17-19

turbinate cells that were tested free of adventitious BVDV. The fetal bovine serum

64

supplement for cell culture medium tested negative for live adventitious BVDV, RNA
from BVDV, and antibody against BVDV. Serial 2-fold dilutions ranging from 1:4 to
1:4,096 were made for each sample of serrun. Antibody titer was considered to be the
highest serum dilution at which the cytopathic effect of the BVDV reference strain was

completely inhibited.

Results

RT-PCR
All skin samples collected fiom all vaccinated cattle in both experiments were

negative for BVDV at all time points using both pooled RT-PCR and individual sample

RT-PCR.

Virus Isolation

In experiment 1, BVDV was not isolated from either serum or nasal swabs. In
experiment 2, BVDV was not isolated fiom nasal swabs, but a total of twenty BVDV
isolates were identified from serum and buffy coat samples. Four of seven animals were
positive on virus isolation fi'om serum on at least one day (n=11) and five of seven were
positive on virus isolation fi'om serum on at least one day (n=9). RT-PCR characterized
18 isolates as BVDV biotype 1 and 2 isolates as BVDV biotype 2. Both BVDV biotype
l and 2 were isolated from 2 of the positive cattle. Results from experiment 2 are
summarized in table 1. A representative sample (n=14) of BVDV isolates from each calf
were selected and sequenced to compare to the vaccine strains as well as to look for any

variation in virus within the individual animal. Genetic sequencing showed that the

65

viruses isolated were 99.9% similar to each other (Figure 3.1). In addition the sequenced
isolates aligned with the type 1 and type 2 viruses contained within vaccine used in the

experiment.

Virus Neutralization

Eleven of 12 calves in experiment 1 had BVDV VN antibodies on day 0 prior to
vaccination and the 12th calf seroconverted by day 18. Conversely, all 7 animals in

experiment 2 were na'r’ve for BVDV antibodies on day 0, and all 7 had seroconverted to
BVDV genotype type 1a and 1b by day 18 and 6 of 7 had converted to BVDV genotype

type 2a by day 18 (Table 2).

Biological Cloning/Sequence Analysis

Biologic cloning of the reconstituted vaccine led to 34 isolates being made. Of
the 34 isolates, RT-PCR amplification led to l isolated being classified as BVDV biotype
1, 25 isolates classified as biotype 2, and 8 were a mix of both biotypes 1 and 2. Genetic
sequencing of the type 1 and type 2 specific isolates showed 99.9% alignment with the
type 1 and type 2 viruses that were reported to be contained within the vaccine (Pfizer
Animal Health, Personal Communication).

A representative sample (n=14) of BVDV isolates fi'om each calf were selected
and sequenced to compare to the vaccine strains as well as to look for any variation in
virus within the individual animal. Genetic sequencing showed that the viruses isolated
were 99.9% similar to each other (Figure 3.1). In addition the sequenced isolates aligned

with the type 1 and type 2 viruses contained within vaccine used in the experiment.

66

Discussion

In this study, all cattle, regardless of their serological status, were negative for
BVDV using individual! and pooled RT-PCR on skin samples following vaccination with
a modified- live BVDV vaccine. These findings indicate that there is a low probability
for detection of vaccine virus in ear notch samples by either individual or pooled RT-
PCR following vaccination with the commercially available BVDV vaccine used in this
study. These findings are similar to previous studies that evaluated skin samples

8,9,11

following vaccination using IHC. In these studies, the authors were unable to detect

BVDV in skin samples from animals that were either acutely infectedg’9 or vaccinated

with BVDV.”

Bovine viral diarrhea virus was not found in any of the samples from calves in
experiment 1. This result was not surprising because of the pre-existing VN antibody
titers present in these calves. In experiment 2, RT—PCR assays (pooled and individual)
did not detect virus in skin samples of vaccinated animals on any of the sample dates.
However, virus isolation from serum and buffy coat, and seroconversion of calves
provides evidence that the vaccine viruses were replicating in the animals during the
testing period. Results from virus isolation of nasal swabs, serum, and buffy coat samples

8,10,20

in experiment 2 were similar to previous findings for seronegative cattle. Reasons

why vaccine virus may not have been detected in skin samples include virulence of the
vaccine strain and location of virus replication. Modified-live vaccines typically are of

reduced virulence and do not replicate to high titers, as compared to virulent field strains

67

of BVDV. It should be noted that when virus isolation was performed on serum and buffy
coats, CPE was often not observed until 5 or more days after cell culture inoculation.

This suggests that there were small amount of viable virus in these samples. If a small
amount of virus exists in the blood, then the probability chances of detecting virus from a
skin sample using any assay including RT-PCR is small. Furthermore the attenuation of
the vaccine may diminish viral replication in vivo in the same sites as field strains of

BVDV might. Previous studies have shown that cytopathic BVDV can be found in the

. . . 2] .
skin of cattle undergorng mucosal disease , however to our knowledge no studies have

looked at distribution of modified-live cytopathic vaccine virus in skin.

One limitation of this study was that only one commercially available modified-
live BVDV vaccine was used. The findings from this study should not be extended to
other modified-live or killed vaccines. In addition, there were no non-vaccinated controls
used in these experiments. Non-vaccinated controls would have served to detect
extraneous virus exposure during the course of the study. However, this limitation was
accounted for by sequencing virus isolated from calves and comparing that sequence
back to the vaccine virus used in the study. The fact that all virus detected were identical
to the viruses present in the vaccine provides substantial evidence that the virus detected
originated from the virus administered.

The results of this study provide evidence that it is unlikely to detect BVDV by
RT-PCR on skin samples following vaccination with a commercially available modified-
live BVDV vaccine. This study supports findings from other studies that have shown

similar results using different assay methods. Veterinarians and producers should feel

68

confident that positive test results for BVDV on skin samples is unlikely to be caused by

vaccination virus following administration of a modified—live virus vaccine.

Sources and Manufacturers

a. Bovi-Shield Gold 5, Pfizer Animal Health, New York, NY

b. TRIzole Reagent, Invitrogen Co., Carlsbad, Calif.

69

Table 3.1 BVDV biotypes isolated in Experiment 2 from Day 7-14

 

 

 

 

 

 

Animal ID
Serum Buffy Coat

Day 82 83 84 86 88 89 91 82 83 84 86 88 89 91
7 - - - la - - - - - - 1a 1a - -
8 - - - 1a - - - - - - 1a 2a - -
9 - - - 1a - - - - - - - - - -
10 - - - 1a - - - - - - - - - -
1 1 - - - - 1a 1a - - - - - - 1a -
12 - - la la - 1a - - - 1a - - 2a 1a
13 - - - 1a - 1a - - - - 1a - - -
14 - - - - - - - - - - - - - -

 

70

 

Table 3.2 Virus Neutralization results at day 0 and day 18 post vaccination with a
commercial modified-live BVDV vaccine (day 0/day18).

 

 

 

 

 

 

 

 

 

VN titers d 0/18
Animal ID Singer 1a TGAC lb 125C 2a
Experiment I
40W 256/32 16/16 64/32
41W 512/512 128/128 256/256
46W 32/32 16/8 32/ 16
50W 4096/2048 1024/512 128/128
904 4/2048 4/128 4/256
906 32/64 4/4 128/64
907 16/128 4/16 16/8
909 32/128 8/32 4/2048
911 2048/512 128/128 256/64
912 128/128 32/16 8/16
914 1024/2048 128/64 1024/256
937 16/ 16 4/4 8/4
Experiment 2

82 <4/256 <4/32 <4/4
83 <4/512 <4/64 <4/128
84 <4/512 <4/128 <4/32
86 <4/64 <4/128 <4/4
88 4/64 <4/ 16 <4/4
89 <4/32 <4/64 <4/64
91 <4/16 <4/<4 <4/4

 

71

 

 

 

 

91 d12 BC

. NADL

89 d12 serum
89 d12 BC

88 d7 BC

84 14 BC

84 d12 serum
Vaccine Type 1
86 d7 serum
86 d7 BC

86 d13 serum
86 d13 BC

88 d11 serum
89 d14 serum
Vaccine Type 2
I 88 d8 BC

 

0.5

|89d11 BC

Figure 3.1 Phylogenetic relationships of 14 BVDV isolates to genotype 1 (Vaccine Type
1) and genotype 2 (Vaccine Type 2) BVDV vaccine used in experiment 2. The neighbor-
joining method was used to generate this phylogenetic tree. The tree is drawn to scale,
with branch lengths in the same units as those of the evolutionary distances used to infer
the phylogenetic tree. The evolutionary distances were computed by use of the Jukes-
Cantor method and are in the units of the number of base substitutions per site.

72

10.

References

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calculating total annual national losses due to bovine viral diarrhoea virus
(BVDV) infection in dairy herds and sensitivity analysis of selected paramenters.
in Proceedings. 2nd Symp.Ruminant Pestiviruses 1993;178-184.

Larson RL. Econonic, reproductive, and performance effects of PI BVD in
commercial cattle operations: managing to minimize losses, in Proceedings. 38'”
Annu Conv Am Assoc Bov Pract 2006;99-106.

Hessman BE, Fulton RW, Sjeklocha DB, et al. Evaluation of economic effects
and the health and performance of the general cattle population after exposure to
cattle persistently infected with bovine viral diarrhea virus in a starter feedlot. Am
J Vet Res 2009;70:73-85.

Wittum TE, Grotelueschen DM, Brock KV, et al. Persistent bovine viral diarrhoea
virus infection in US beef herds. Prev Vet Med 2001;49:83-94.

Sandvik T. Selection and use of laboratory diagnostic assays in BVD control
programmes. Prev Vet Med 2005;72:3-16.

Grooms DL, Brock KV, Ward LA. Detection of bovine viral diarrhea virus in the
ovaries of cattle acutely infected with bovine viral diarrhea virus. J Vet Diagn
Invest 1998;10:125-129.

Givens MD, Riddell KP, Zhang Y, et al. Safety and efficacy of vaccination of
seronegative bulls with modified-live, cytopathic bovine viral diarrhea viruses.
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SUMMARY

Sentinel serology is a usefirl method for detecting the presence of BVDV in cattle
herds. This strategy appears to be an effective and economically viable secondary test for
follow-up testing in herds that have completed whole herd testing previously. In the case
of an eradication program, it is important to develop inexpensive means of surveillance
testing that allows for on-going herd monitoring. In a voluntary eradication program,
having a cost-effective test available will aid producers in continuing to monitor their
herd without a significant financial burden. If a mandatory government program is in
place, sentinel testing also fits in as an excellent surveillance tool that is easy to
implement and economically feasible, especially when financial resources are limited.

In addition, at the herd level, knowing if a herd has been re-infected with BVDV
will allow veterinarians and producers to modify biocontainment, biosecurity, and
vaccination protocols. Likewise, knowing that a herd is still BVDV-free, confirms that
any control protocols irriplemented appear to be working. It may also allow for herd
owners to market their animals as BVDV free. From a regional standpoint, it will allow
veterinarians to isolate and research any BVDV positive herds to determine how the
disease was re-introduced to the herd so that spread of the disease can be controlled and
prevented. The ability to identify where the disease came from will allow veterinarians
and producers alike to take the proper precautions to prevent re-introduction of the virus
into additional herds.

In the face of a positive BVDV results, producers could be faced with costs
associated with firrther individual or herd testing or more significantly, culling of

suspected PI cattle. Therefore, false positive test results can be very costly to the

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producer. As newer and more sensitive diagnostic tests are deve10ped, it is important to
conduct studies such as the one reported in this thesis, in order to confidently interpret the
results. BVDV vaccine virus could not be detected in skin following vaccination with a
modified live BVDV vaccine. When coupled with findings of previous studies, this
suggests that it is unlikely to detect BVDV by RT-PCR on skin samples following
vaccination. Thus, veterinarians should be able to confidently tell producers that recent
vaccination of cattle with a MLV BVDV vaccine should not lead to false positive results.
The vaccine study ties into the eradication of BVDV from a specific region in the
sense that by knowing that it is highly unlikely to find vaccine virus in the skin samples
of cattle. Therefore veterinarians and producers should feel confident that positive test
results for BVDV on skin samples is unlikely to be caused by vaccination virus following
administration of a modified-live virus vaccine. Positive BVDV results from any form of
testing should serve as a red flag that BVDV exists within that herd and steps should be

taken to isolate and eliminate any animals with the virus.

The Future

In a perfect world, the sentinel serology study could be repeated with a larger
sample size and using herds with both known and unknown BVDV status. Having a
greater number of herds with BVDV present would allow for sensitivity and specificity to
be more accurately measured. However it should be stressed that the goal of this
particular study was to evaluate the sentinel serology in an eradication project, not to
determine the sensitivity and specificity of the test. In addition, since this test is being

evaluated as a follow-up surveillance tool, it would be interesting to conduct this study

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over several years. By doing so, producers would be able to see if their biosecurity and
vaccination protocols have prevented the re-introduction of BVDV into their herds, as
well as determining if sentinel serology works as a regional surveillance too]. Of course
it should be noted that there are always exceptions to the rule. Many herds exist where
sentinel serology may prove to be impractical. One such example is any herd where
vaccination programs begin within the first weeks or months of a calf’ 8 life. In herds
such as this, sentinel serology would be less practical and other screening modalities
would need to be explored. One way to overcome this problem would be to leave 5
calves unvaccinated until sentinel testing occurs or by placing a separate sentinel species,
such as sheep, within the herd.

Regarding the vaccination study, to the most important way to build upon this
study would be to evaluate different vaccines on the market. In addition to other
modified-live cytopathic vaccines, evaluation of modified-live non-cytopathic vaccines,
as well as killed vaccines would be of value. By doing this, one would be able to more
confidently state that vaccine virus cannot be detected in skin samples using RT-PCR
following vaccination.

In summary, our research has advanced cattle health and well-being by further
demonstrating the usefulness of sentinel serology as a tool for controlling and preventing
BVDV in cattle, specifically in its application in a BVDV eradication program in North
America. Furthermore, we have provided strong evidence that modified live BVDV
vaccines are unlikely to cause false positive results on common BVDV detection assays,

thus simplifying the interpretation of these test results.

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