K. V. Brock and V. S. Cortese

Experimental Fetal Challenge Using Type II Bovine Viral Diarrhea Virus in Cattle Vaccinated With Modified-Live Virus Vaccine* Kenny V. Brock, DVM, MS, PhDa Victor S. Cortese, DVM, PhDb

aAuburn

University College of Veterinary Medicine Department of Pathobiology Auburn, AL 36849

bPfizer

Animal Health 812 Springdale Drive Exton, PA 19341

■ ABSTRACT Nineteen open heifers or cows were vaccinated 45 days prior to breeding with a modified-live bovine viral diarrhea virus (BVDV) vaccine. An additional six animals were not vaccinated and served as controls. All 25 animals were estrus-synchronized and bred. At 75 days of gestation, the 25 pregnant animals were experimentally infected with a type II isolate of BVDV by intranasal inoculation. At 75 days after inoculation, the animals were euthanized and each fetus was removed and retained for sampling. Virus isolation was accomplished from fetal tissues (spleen, thymus, and small intestine). Type II BVDV was isolated from the fetuses collected from all six unvaccinated control animals and from eight of 19 fetuses from vaccinated animals, which were determined to be persistently infected following experimental challenge. *This study was supported in part by Pfizer Animal Health, New York, NY.

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■ INTRODUCTION The prevention of bovine viral diarrhea virus (BVDV) disease must focus on the prevention of persistently infected animals. The identification and removal of persistently infected animals and vaccination are the basis for effective control methods. Bovine viral diarrhea virus has adapted to the bovine by a unique ability to cross the placenta and infect the developing fetus. Although there may be several consequences to infection in utero, the development of persistent infections in the fetus following transplacental infection with a noncytopathic strain of BVDV up to approximately 125 days of gestation is recognized as a major problem in the control of this disease.1 Currently, the mechanism(s) of transplacental transfer of BVDV is unknown and there is little information regarding the protective mechanisms that are important for the prevention of fetal infections.2 Although dams have measurable levels of anti-BVDV antibody, a sufficient amount of virus is able to escape inactivation and cause

Veterinary Therapeutics • Vol. 2, No. 4, Fall 2001

transplacental infection and the development of persistent fetal infections.2–4 It is essential that vaccination provide fetal protection to eliminate the insidious problem of infections in utero that lead to the birth of persistently infected calves. Several studies have been performed to assess the ability of vaccines to protect the fetus against either natural or artificial challenge.4–6 Brownlie and coworkers5 recently reported on the efficacy of an inactivated BVDV vaccine for prevention of fetal infection. To date, vaccines licensed in the United States have not been required to demonstrate fetal protection; however, there is current interest in determining the ability of BVDV vaccines to provide this level of protection. Efficacy of some existing vaccines has been reevaluated recently, subsequent to the recognition of genetic and antigenic diversity of BVDV isolates.7,8 Previously, using an experimental fetal challenge model with type I BVDV, a modifiedlive virus vaccine provided protection against the development of persistent infection in 88% of vaccinated pregnant heifers.4 The purpose of the present study was to determine the ability of a modified-live vaccine to provide fetal protection against challenge with a type II isolate of BVDV, using similar experimental conditions as were used for a type I BVDV vaccine challenge study. ■ MATERIALS AND METHODS Animals and Vaccinations Twenty-five open heifers and cows were used in the study. Six cattle were kept as nonvaccinated controls, and 19 animals were vaccinated with a modified-live vaccine (Resvac® 4/Somnubac®; Pfizer Animal Health, New York, NY). All animals were determined to be negative for BVDV by virus isolation from serum and whole blood and by polymerase chain reaction (PCR) from RNA extracted

from whole blood prior to initiation of the study. The vaccine (2.0 ml) was administered by intramuscular injection in the neck of each assigned animal. Blood samples were collected from all animals for serology at the time of vaccination and prior to experimental challenge. Breeding Forty-five days following vaccination, all cattle were given prostaglandin F2α (Lutalyse®, Pharmacia-Upjohn, Kalamazoo, MI) and exposed to 10 bulls that were BVDV-negative by virus isolation and were seronegative for BVDV antibody. The first breeding exposure was for one week. Animals were examined for pregnancy status by ultrasound at Day 30 following the first day of exposure. Following ultrasound examination, all open animals were given a second dose of prostaglandin F2α and then exposed to the bulls for another 7 days, followed by another ultrasound to determine pregnancy status. Virus Challenge Experimental challenge was conducted in two groups based on pregnancy status. The first group consisted of 19 animals and the second group contained six animals; each group contained both vaccinates and at least one control. Challenge was carried out by intranasal administration of 5 ml of inoculum through an aerosolizer. The challenge dose consisted of a total of 5.0 × 105 cell culture infective dose (CCID)50 of the PA131 strain of a noncytopathic type II BVDV. The inoculum was made from a single stock with a titer of 107 CCID50/ml. The PA131 type II BVDV was isolated by the investigator from a persistently infected bull in Pennsylvania. Evaluations Nasal swabs and whole-blood samples were collected for virus isolation on the day of chal-

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K. V. Brock and V. S. Cortese

lenge and 3, 4, 5, 6, 7, 8, 10, 14, and 30 days after challenge. Body temperatures also were recorded on these days. Animals were observed each day for clinical signs of disease such as diarrhea, anorexia, nasal discharge, increased rate of respiration, or general malaise. Following challenge, all animals were examined by ultrasound at weekly intervals for 4 weeks to determine fetal viability by observation of a fetal heartbeat. Fetus Evaluations At approximately 150 to 180 days of gestation, all dams were euthanized and the spleen, thymus, and small intestine were collected from each fetus. Sections of each tissue were harvested for virus isolation. Tissue samples were homogenized for 5 minutes in 2 ml of Dulbecco’s Modification of Eagle’s Medium using a commercial circulator/stirrer. Virus isolation from tissue homogenates was accomplished by performing two passages on bovine turbinate cells with detection of viral antigen by immunoperoxidase assay.9 Polymerase chain reaction was completed from RNA extracted from the tissue homogenates10,11 and PCR-typing was done according to procedures described by Ridpath and coworkers8 from RNA extracted from cell culture supernatants that were positive by immunoperoxidase assay. Serology was done by standard serum virus neutralization assay using the type I (NADL) and the type II virus

(PA131).12 The NADL and PA131 BVDV isolates correspond to the vaccine strain and the challenge strain, respectively. Statistical Analysis Data between the two groups (vaccinates and nonvaccinates) were compared within each challenge group and between the two groups with the two challenge groups combined using chi-square analysis. Differences were declared significant when P < .05. ■ RESULTS Following experimental challenge of the 25 pregnant animals, BVDV was isolated from blood samples collected from five of six unvaccinated dams (Table 1). There was no virus isolation from any nasal swabs collected. During the period immediately following challenge, BVDV was not isolated from any of the 19 animals that previously had been vaccinated. For 14 days following experimental infection, no clinical signs were observed from any of the experimentally infected animals. Serology was negative (1:640 1:544 1:36