Mary L. Vickers

Quantitation of bovine viral diarrhea virus in embryo transfer flush fluids collected from a persistently infected heifer

Due to the epidemiological significance of persistently infected animals, it is important that possible methods of transmission be investigated. Currently, the recommended prevention and control measures for BVDV are centered around the identification and removal of persistently infected animals and the judicious use of BVDV vaccines. 4,6 Unfortunately, the identification of persistently infected animals has not been emphasized until recently and remains impractical. The purpose of this report is to emphasize the epidemiological significance of animals persistently infected with BVDV, especially in relation to embryo transfer procedures. A 5-month-old Holstein heifer, persistently infected with BVDV, was identified in a dairy herd with a history of BVDV infection by virus isolation from nasal and vaginal swabs and serum. The heifer had no detectable levels of BVDV serum antibodies (in indirect fluorescence assay [IFA]) and was kept in isolation for observation and testing. Estrus first was detected at 14 months of age. Following the third heat cycle the heifer was superovulated with follicle-stimulating hormone (days 11-14) and inseminated with BVDV-negative semen (confirmed negative by virus isolation) during the subsequent estrous. Embryo collection was attempted on day 7 (estrus day 0) by standard whole body uterine flush using 2 liters of Dulbecco’s phosphate-buffered saline containing 3% BVDV-negative fetal bovine serum (confirmed negative by virus isolation). Virus was not isolated from the flush medium prior to the uterine flush. Only 3 unfertile eggs were identified by microscopic examination in the uterine flush fluid (2 liters). The titers of BVDV were quantitated in various samples collected from the persistently infected heifer by virus isolation from 10-fold dilutions of the samples. One milliliter of each dilution was inoculated in replicates of 3 onto BVDVnegative secondary bovine turbinate cells passaged 5-10 times in Dulbecco’s minimum essential medium containing 10% horse serum. The inoculum was removed after 1 hour and

The Increased Prevalence of Neuropathogenic Strains of EHV-1 in Equine Abortions

A panel of 426 archived EHV-1 isolates collected (1951-2006) from equine abortions was analyzed using a real-time Taq-Man((R)) allelic discrimination PCR assay. Based on previous findings, isolates possessing adenine at nucleotide position 2254 (A(2254)) in ORF30 were classified as having a non-neuropathogenic genotype and those with guanine at 2254 (G(2254)) were designated as the neuropathogenic genotype. The resultant data demonstrated that viruses with the neuropathogenic genotype existed in the 1950s and isolates with this genotype increased from 3.3% in the 1960s to 14.4% in the 1990s. The incidence of EHV-1 isolates from 2000 to 2006 with G at position 2254 is 19.4%, suggesting that viruses with the neuropathogenic genotype are continuing to increase in prevalence within the latent reservoir of the virus, leading to greater risks for costly outbreaks of equine herpesvirus neurologic disease. Another highly significant finding was two isolates failed to react with either probe in the allelic discrimination assay. These isolates were found to possess an adenine to cytosine substitution at position 2258 (A(2258)-->C(2258)) in ORF30, in addition to A(2254)-->G(2254). Interestingly, the non-neuropathogenic RAC-H modified live vaccine strain of EHV-1 also contains both A(2254)-->G(2254) and A(2258)-->C(2258) substitutions. This finding clearly suggests that additional research is required before the genetic basis of the neuropathogenic phenotype in EHV-1 is fully understood.

Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses.

The orthoreoviruses are segmented RNA viruses that infect diverse vertebrate host species. While the most common human orthoreovirus, Mammalian Reovirus, is not typically associated with significant disease, the majority of Orthoreovirus species have been shown to cause significant and often fatal disease in reptiles, birds, and primates. There is significant potential for jumping species. A consensus nested-PCR method was designed for investigation of the RNA-dependent RNA polymerase gene of Orthoreovirus and Aquareovirus. This protocol was used to obtain sequencing template from reoviruses of three different vertebrate classes. Bayesian and maximum likelihood phylogenetic analysis found that all viruses analyzed clustered in the genus Orthoreovirus, that reptile reoviruses formed three distinct clusters, and that an African grey parrot reovirus clustered with Nelson Bay virus from bats. This PCR method may be useful for obtaining templates for initial sequencing of novel orthoreoviruses from diverse vertebrate hosts.

Evaluation of a Human Group A Rotavirus Assay for On-Site Detection of Bovine Rotavirus

ABSTRACT Neonatal diarrhea induced by bovine group A rotavirus causes significant economic loss in the dairy and beef industry due to increased morbidity and mortality, treatment costs, and reduced growth rates. The objective of this study was to evaluate a human group A rotavirus assay (ImmunoCardSTAT Rotavirus [ICS-RV]) as an on-site diagnostic test for bovine rotavirus. When used with a collection of bovine diarrhea samples submitted to the Virology Section of the Diagnostic Center for Population and Animal Health at Michigan State University and compared to a bovine group A rotavirus-specific reverse transcription-PCR (RT-PCR), the ICS-RV assay had a sensitivity and specificity of 87.0 and 93.6%, respectively. A commercially available group A rotavirus enzyme-linked immunosorbent assay (ELISA) (Pathfinder; Sanofi Diagnostics, Redmond, Wash.), when used with the same fecal sample collection and compared to the same RT-PCR, had a sensitivity and specificity of 78.3 and 67.7%, respectively. Subsequently, the ICS-RV assay, RT-PCR, and a different commercially available group A rotavirus ELISA (Rotaclone; Meridian Diagnostics, Cincinnati, Ohio) were used to evaluate fecal samples collected from neonatal calves experimentally infected with bovine rotavirus. When diarrheic fecal samples that were positive for bovine rotavirus by RT-PCR were evaluated, the ICS-RV assay and the Rotaclone assay detected bovine rotavirus 85 and 95% of the time, respectively. Based on these studies, the ICS-RV assay appears to be an excellent test for detecting group A bovine rotaviruses. This assay may be useful as an on-site diagnostic test for veterinarians as an aid in the management of bovine neonatal diarrhea.

Development and Evaluation of One-Step TaqMan Real-Time Reverse Transcription-PCR Assays Targeting Nucleoprotein, Matrix, and Hemagglutinin Genes of Equine Influenza Virus

ABSTRACT The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Microbiologic and pathologic findings in an epidemic of equine pericarditis

During the spring and summer of 2001 and in association with the mare reproductive loss syndrome, 22 terminal and 12 clinical cases of equine pericarditis were diagnosed in central Kentucky. Actinobacillus species were the principal isolates from 8 of 10 nontreated, terminally affected and 3 of 10 clinically affected horses. Enterococcus faecalis and Streptococcus zooepidemicus were cultured from the remaining 2 nontreated terminal cases. No viruses were isolated in tissue culture. Nucleic acid of equine herpesvirus-2 was detected in pericardial and tracheal wash fluids of 3 and 1 individuals, respectively. Microscopic alterations in sections of heart and parietal pericardium were consistent with chronic fibrinous bacterial pericarditis. This report confirms a significant role of Actinobacillus species in equine pericarditis and describes an epidemic of this infrequently observed syndrome in the horse.

Abortion and ulcerative posthitis associated with caprine herpesvirus-1 infection in goats in California.

Three outbreaks of late-gestation abortions in does and ulcerative posthitis in bucks, associated with caprine herpes virus–1 (CHV-1), in California are described. In herd A, 10 of 17 does aborted in a 7-day period, whereas in herd B, 4 of 130 does aborted in a 45-day period and in herd C, 100 of 300 does aborted in a 3-week period. Most fetuses had multifocal pinpoint depressed foci with a zone of hyperemia on external and cut surfaces of the kidneys, liver, lungs, and adrenal glands. Histologically, scattered multifocal areas of necrosis with mild neutrophilic infiltrate were observed in kidneys, brain, liver, adrenal glands, and lungs of most fetuses of the 3 herds. Large amphophilic intranuclear inclusion bodies, which displaced the chromatin, were observed in cells within and around the necrotic foci in kidneys and adrenal glands. Particles 85–113 nm in size with morphology compatible with herpes virus were observed in the nuclei of these cells when examined by electron microscopy. Irregular, shallow, red ulcers were observed in the prepuce of 1 buck from herd C. Prepuce biopsies from this animal had necrosis of the superficial mucosal epithelium and severe submucosal lymphoplasmocytic infiltrates. Large intranuclear amphophilic inclusion bodies were observed in most cells of the stratum spinosum of the preputial epithelium, but no viral particles were observed in these cells. Caprine herpes virus–1 was isolated from tissue pools of fetuses from the 3 herds but not from prepuce biopsies. Positive results were obtained when tissues of a fetus from herd C were processed by a polymerase chain reaction technique to amplify the amino terminus of the glycoprotein C gene of CHV-1. Sera from aborted does from herds B and C and from the 3 bucks from herd C had high antibody titers to CHV-1. The results presented here support the hypothesis that the male goat is involved in the transmission of CHV-1. However, other forms of transmission cannot be ruled out.

Equine abortion and premature birth associated with Cellulosimicrobium cellulans infection

During the 2002 and 2003 foaling seasons, Cellulosimicrobium (Cellumonas) cellulans (formerly Oerskovia xanthineolytica) was the principal microorganism isolated from fetal tissues or placentas from cases of equine abortion, premature birth, and term pregnancies. Significant pathologic findings included chronic placentitis and pyogranulomatous pneumonia. In addition, microscopic and macroscopic alterations in the allantochorion from 4 of 7 cases of placentitis were similar to those caused by Crossiella equi and other nocardioform bacteria. This report confirms a causative role of C. cellulans infection in equine abortion.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

ABSTRACT The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP51-255], M1-162, and N1-110), as well as partial sequences of these structural proteins (GP51-116, GP575-112, GP555-98, M88-162, and N1-69) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP555-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP555-98 MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP555-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

Dermatophilus congolensis-associated placentitis, funisitis and abortion in a horse

Placentitis, funisitis and fetal bronchopneumonia were diagnosed in an aborted full-term Thoroughbred fetus and its placenta by histopathological examination. Dermatophilus congolensis organisms were isolated from placenta, lung and stomach content. The genotypic identification of aerobic culture was confirmed by sequential analysis of the entire 16S rDNA gene. This is the first report of Dermatophilus congolensis-associated abortion in any species.