Whole-genome sequences of Odocoileus hemionus deer adenovirus isolates from deer, moose and elk are highly conserved and support a new species in the genus Atadenovirus.

We present the first complete genome sequence of Odocoileus hemionus deer adenovirus 1 (OdAdV-1). This virus can cause sporadic haemorrhagic disease in cervids, although epizootics with high mortality have occurred in California. OdAdV-1 has been placed in the genus Atadenovirus, based on partial hexon, pVIII and fibre genes. Ten field isolates recovered from naturally infected mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginiana) and moose (Alces alces) from Wyoming, black-tailed deer (Odocoileus hemionus columbianus) from California, and Rocky Mountain elk (Cervus elaphus nelsoni) from Colorado and Washington state were sequenced. The genome lengths ranged from 30 620 to 30 699 bp, contained the predicted proteins and gene organization typical of members of genus Atadenovirus, and had a high percentage of A/T nucleotides (66.7 %). Phylogenic analysis found that the closest ancestry was with ruminant atadenoviruses, while a divergence of the hexon, polymerase and penton base proteins of more than 15 % supports classification as a new species. Genetic global comparison between the 10 isolates found an overall 99 % identity, but greater divergence was found between those recovered from moose and elk as compared to deer, and a single variable region contained most of these differences. Our findings demonstrate that OdAdV-1 is highly conserved between 10 isolates recovered from multiple related cervid species, but genotypic differences, largely localized to a variable region, define two strains. We propose that the virus type name be changed to cervid adenovirus 1, with the species name Cervid atadenovirus A. Sequence data were used to develop molecular assays for improved detection and genotyping.

Culiseta annulata: A New Mosquito For Kuwait.

We report new records for Culiseta annulata from Kuwait. Prior to our records, Culiseta longiareolata was the only Culiseta sp. known from Kuwait. Culiseta annulata is a vector of Tahyna virus (Bunyaviridae) to humans throughout Asia. We tested a limited number of mosquitoes for Tahyna virus and other viruses. Tahyna virus was not detected, but we did discover a mosquito Densovirus in a pool of Cs. annulata using next generation sequencing.

Laboratory Validation of the Sand Fly Fever Virus Antigen Assay.

Sandfly fever group viruses in the genus Phlebovirus (family Bunyaviridae) are widely distributed across the globe and are a cause of disease in military troops and indigenous peoples. We assessed the laboratory sensitivity and specificity of the Sand Fly Fever Virus Antigen Assay, a rapid dipstick assay designed to detect sandfly fever Naples virus (SFNV) and Toscana virus (TOSV) against a panel of phleboviruses. The assay detected SFNV and TOSV, as well as other phleboviruses including Aguacate, Anahanga, Arumowot, Chagres, and Punta Toro viruses. It did not detect sandfly fever Sicilian, Heartland, Rio Grande, or Rift Valley fever viruses. It did not produce false positive results in the presence of uninfected sand flies (Lutzomyia longipalpis) or Cache Valley virus, a distantly related bunyavirus. Results from this laboratory evaluation suggest that this assay may be used as a rapid field-deployable assay to detect sand flies infected with TOSV and SFNV, as well as an assortment of other phleboviruses.

Culicoides sonorensis (Diptera: Ceratopogonidae) is not a competent vector of Cache Valley virus (family Bunyaviridae, genus Orthobunyavirus).

We investigated the susceptibility of Culicoides sonorensis to Cache Valley virus (CVV) (family Bunyaviridae, genus Orthobunyavirus) infection and the potential that it could be a vector or site of virus reassortment. CVV is native to the New World and causes disease in livestock. Infected blood meals were fed to both a competent vector, Anopheles quadrimaculatus, and Culicoides sonorensis. All Anopheles mosquitoes were infected as expected, but only 21 % of the C. sonorensis insects were susceptible to infection. These appeared to present a midgut barrier, because virus persisted but did not disseminate. This means Culicoides sonorensis is not likely to be a vector of CVV but could be involved in viral reassortment. Schmallenberg virus (SBV) (family Bunyaviridae, genus Orthobunyavirus) was recently discovered in Europe and probably is a novel virus resulting from a reassortment of two orthobunyaviruses, and an ongoing epizootic in cattle and small ruminants has caused significant economic damage.

Adenovirus Hemorrhagic Disease in Moose (Alces americanus gigas) in Alaska, USA.

In 1993, an epizootic of adenovirus hemorrhagic disease (AHD) caused the death of at least 1,000 mule deer (Odocoileus hemionus) in California, US. Since then, numerous cervid species throughout the US have had deaths confirmed to be caused by AHD. In 2015, the death of two captive moose (Alces americanus gigas) calves marked the first recognized AHD-caused deaths in Alaska, a state in which moose are important economically as well as for food security and cultural identity. Both cases were characterized by systemic vasculitis with endothelial cell intranuclear inclusion bodies, pulmonary edema, petechial hemorrhages, and enterotyphlocolitis.

Further Characterization of Rio Grande Virus and Potential for Cross Reactivity with Rift Valley Fever Virus Assays.

Phleboviruses (genus Phlebovirus, family Phenuiviridae) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep (Ovis aries) and horses (Equus caballus) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.

Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

Investigation of a bluetongue disease epizootic caused by bluetongue virus serotype 17 in sheep in Wyoming.

OBJECTIVE: To characterize a 2007 bluetongue disease (BT) epizootic caused by bluetongue virus (BTV) serotype 17 in sheep in the Big Horn Basin of Wyoming. DESIGN: Cross-sectional study. ANIMALS: 1,359 sheep from ranches in Wyoming and Montana. PROCEDURES: Information on clinical signs and history of BT in sheep was obtained from ranchers and attending veterinarians. At 3 to 6 months after the 2007 BT epizootic, blood samples were collected from rams, ewes, and lambs within and outside the Big Horn Basin; blood samples were also collected from lambs born in the spring of 2008. Sera were tested for anti-BTV antibodies by use of a competitive ELISA to determine the seroprevalence of BTV in sheep and to measure antibody titers. Virus isolation and reverse transcriptase PCR assays were used to determine long-term presence of the infectious virus or viral genetic material in RBCs of sheep. RESULTS: The percentage of sheep seropositive for BTV closely matched morbidity of sheep within flocks, indicating few subclinical infections. Flocks separated by as little as 1 mile had substantial variation in infection rate. Rams were infected at a higher rate than ewes. There was no evidence of BTV successfully overwintering in the area. CONCLUSIONS AND CLINICAL RELEVANCE: This epizootic appears to be a new intrusion of BTV into a naïve population of sheep previously protected geographically by the mountains surrounding the Big Horn Basin. Rams may have a higher infection rate as a result of increased vector biting opportunity because of the large surface area of the scrotum.

Aqueous 2% geraniol as a mosquito repellent failed against Aedes aegypti on ponies.

Organic insect repellents are of interest to many agricultural producers and animal owners. Geraniol, a plant-derived alcohol, is naturally produced by a wide range of plants and is a US Environmental Protection Agency minimum risk pesticide. Previous studies have shown various concentrations of geraniol repel or kill mosquitoes; however, geraniol might cause allergic contact dermatitis in humans or animals. We tested a commercially available 2% aqueous solution of geraniol on ponies as a mosquito repellent. Five trials were conducted on ponies treated with a 60-ml aerosol mist (30 ml per side) of 2% geraniol or as untreated controls. Animals were observed 3 h postapplication to check for skin irritation. Aedes aegypti, in feeding tubes, were held on the ponies for 7 min. The average percent of biting on control animals was 56%, with a range of 16-90%, and the average for the treatments was 13%, with a range of 0-86%. Based on statistical models, there was no significant difference (P = 0.081) in the percent bites between treated and untreated animals after 3 h. Based on our data, 2% geraniol was not an adequate mosquito repellent for horses. We did not observe any skin irritation on the animals treated with 2% geraniol.

Rickettsia hoogstraalii and a Rickettsiella from the Bat Tick Argas transgariepinus, in Namibia.

Ectoparasites were collected from Eptesicus hottentotus, the long-tailed serotine bat, caught in Namibia as part of an ecological study. Larvae of Argas transgariepinus, a blood-feeding ectoparasite of bats in Africa, were removed from 3 of 18 bats. We present scanning electron microscope images of unengorged larvae. As with other ectoparasites, this bat tick might transmit pathogens such as Borrelia and Rickettsia to their hosts as has been reported for bat ticks in Europe and North America. We screened 3 pools (25 total) of larvae of A. transgariepinus removed from the long-tailed serotine bat Eptesicus hottentotus caught in Namibia. Two microbes of unknown pathogenicity, including Rickettsia hoogstraalii, a spotted fever group pathogen, and a Rickettsiella sp. were detected by molecular techniques.

Maternal obesity accelerates fetal pancreatic beta-cell but not alpha-cell development in sheep: prenatal consequences.

Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

New National Record for Culex perexiguus from Kuwait.

Mosquitoes can transmit a wide variety of viral and parasitic pathogens. Several species have recently been reported in new locations throughout the Arabian Peninsula as a result of entomological surveillance by the US military. We report a new national record for Culex perexiguus from Kuwait based on morphologic and molecular identification of captured samples. This mosquito might pose a public health threat to local populations and to military personnel as a potential vector of both Sindbis and West Nile viruses.

Bovine abortion caused by Coxiella burnetii: report of a cluster of cases in Uruguay and review of the literature.

A cluster of 4 bovine abortions caused by Coxiella burnetii occurred in a dairy herd in Uruguay during a 2-mo period. Case 1 consisted of a placenta from an aborted cow; cases 2-4 were fetuses and their placentas. Grossly, the placenta from one aborted cow had moderate, diffuse reddening of the cotyledons and loss of translucency of the intercotyledonary areas. No gross lesions were observed in the other 3 placentas. Microscopically, 2 of 4 placentas had fibrinonecrotizing placentitis with abundant intratrophoblastic gram-negative coccobacilli. C. burnetii was identified intralesionally by immunohistochemistry (IHC) in all 4 placentas, and by PCR and DNA sequencing in 3 placentas analyzed by these techniques. One fetus had mild neutrophilic alveolitis with multinucleate syncytial cells; no gross or microscopic lesions were observed in the other 2 fetuses examined. The lungs of the 3 fetuses were negative for C. burnetii by IHC. Tests performed to investigate other possible causes of abortions in the 4 cases were negative. C. burnetii causes Q fever in humans and coxiellosis in animals. Clusters of abortions in cattle by C. burnetii have not been reported previously, to our knowledge; this bacterium has been considered an opportunistic pathogen associated only with sporadic abortion in cattle. We present herein a cluster of 4 bovine abortions caused by C. burnetii in a dairy farm during a period of 2 mo and a review of the literature on C. burnetii infection in cattle.

Infectious keratoconjunctivitis in free-ranging mule deer in Wyoming: a retrospective study and identification of a novel alphaherpesvirus.

We describe the clinicopathologic findings, relative prevalence, and pathogens associated with infectious keratoconjunctivitis in mule deer ( Odocoileus hemionus) in Wyoming. Seventeen cases with ocular lesions were identified among 1,036 mule deer postmortem submissions (1.6%) in an ~16 y period. Sixteen cases were observed in winter and most were in male (15 cases) and juvenile (13 cases) deer. Blindness was the most commonly reported clinical sign (10 cases). A herpesvirus was detected only in the 4 cases of bilateral necrotizing bulbar conjunctivitis. Phylogenetic analysis of glycoprotein amino acid sequences consistently identified this virus as a novel alphaherpesvirus. In 2 of these herpesvirus-positive cases, Actinomyces sp. and Moraxella ovis were also identified. Trueperella pyogenes was identified in 4 cases of unilateral ulcerative keratitis, keratoconjunctivitis, and panophthalmitis. M. ovis was cultured from 3 cases of bilateral conjunctivitis and keratoconjunctivitis. In the remaining cases, isolates included Moraxella bovis (1 case), Staphylococcus sp. and Streptococcus sp. (2), Flavobacterium sp. and Pseudomonas sp. (2), Escherichia coli and Enterobacter sp. (1), and bovine viral diarrhea virus 1 (1). No pathogens were identified in 2 cases. The relative prevalence of keratoconjunctivitis in mule deer in Wyoming appears to be low, and this disease is most commonly associated with infection by a novel alphaherpesvirus, T. pyogenes, and M. ovis.

Operational mosquito and vector-borne diseases surveillance at Incirlik Air Base, Turkey.

Arboviruses on Incirlik Air Base, Turkey, pose a threat to military personnel and civilians, but might also be relevant for understanding the threats in neighboring conflict zones such as Syria. We reviewed 6 years of mosquito and arbovirus surveillance at Incirlik Air Base. Over 6,000 mosquitoes were identified as Aedes caspius, Anopheles claviger, Culex mimeticus, Cx. perexiguus, Cx. pipiens, Cx. sinaiticus, and Culiseta longiareolata. Almost all of the mosquitoes (more than 90%) were Cx. perexiguus or Cx. pipiens. Both West Nile virus and Sindbis virus were detected in 6 mosquito pools among collections made in 2013, 2014, and 2015.

Expected Net Benefit of Vaccinating Rangeland Sheep against Bluetongue Virus Using a Modified-Live versus Killed Virus Vaccine.

Recurring outbreaks of bluetongue virus in domestic sheep of the US Intermountain West have prompted questions about the economic benefits and costs of vaccinating individual flocks against bluetongue (BT) disease. We estimate the cost of a BT outbreak on a representative rangeland sheep operation in the Big Horn Basin of the state of Wyoming using enterprise budgets and stochastic simulation. The latter accounts for variability in disease severity and lamb price, as well as uncertainty about when an outbreak will occur. We then estimate the cost of purchasing and administering a BT vaccine. Finally, we calculate expected annual net benefit of vaccinating under various outbreak intervals. Expected annual net benefit is calculated for both a killed virus (KV) vaccine and modified-live virus vaccine, using an observed price of $0.32 per dose for modified-live and an estimated price of $1.20 per dose for KV. The modified-live vaccine's expected annual net benefit has a 100% chance of being positive for an outbreak interval of 5, 10, or 20 years, and a 77% chance of being positive for a 50-year interval. The KV vaccine's expected annual net benefit has a 97% chance of being positive for a 5-year outbreak interval, and a 42% chance of being positive for a 10-year interval. A KV vaccine is, therefore, unlikely to be economically attractive to producers in areas exposed less frequently to BT disease. A modified-live vaccine, however, requires rigorous authorization before legal use can occur in Wyoming. To date, no company has requested to manufacture a modified-live vaccine for commercial use in Wyoming. The KV vaccine poses less risk to sheep reproduction and less risk of unintentional spread, both of which facilitate approval for commercial production. Yet, our results show an economically consequential tradeoff between a KV vaccine's relative safety and higher cost. Unless the purchase price is reduced below our assumed $1.20 per dose, producer adoption of a KV vaccine for BT is likely to be low in the study area. This tradeoff between cost and safety should be considered when policymakers regulate commercial use of the two vaccine types.

Comparison of the humoral response between sheep vaccinated with a killed-virus vaccine and those vaccinated with a modified-live virus vaccine against bluetongue virus serotype 17.

OBJECTIVE To compare the humoral response between sheep vaccinated with a killed-virus (KV) vaccine and those vaccinated with a modified-live virus (MLV) vaccine against bluetongue virus (BTV) serotype 17. DESIGN Randomized clinical trial followed by a field trial. ANIMALS 30 yearling crossbred ewes (phase 1) and 344 sheep from 7 Wyoming farms (phase 2). PROCEDURES In phase 1, ewes seronegative for anti-BTV antibodies received sterile diluent (control group; n = 10) or an MLV (10) or KV (10) vaccine against BTV-17 on day 0. Ewes in the KV group received a second dose of the vaccine on day 21. Ewes were bred 5 months after vaccination and allowed to lamb. Anti-BTV antibodies were measured in ewes at predetermined times after vaccination and in their lambs once at 5 to 10 days after birth. In phase 2, 248 commercial sheep were screened for anti-BTV antibodies and vaccinated with a KV vaccine against BTV-17 on day 0. Sheep seronegative for anti-BTV antibodies on day 0 (n = 90) underwent follow-up serologic testing on day 365 along with 96 unvaccinated cohorts (controls). RESULTS In phase 1, all vaccinated ewes developed anti-BTV antibodies by 14 days after vaccination and remained seropositive for 1 year; all of their lambs were also seropositive. All control ewes and lambs were seronegative. In phase 2, the prevalence of vaccinated sheep with anti-BTV antibodies 1 year after vaccination was 93% and 76% as determined by a serum neutralization assay and competitive ELISA, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Both vaccines induced antibodies against BTV-17 that persisted for at least 1 year and provided passive immunity for lambs and may be a viable option to protect sheep against disease.

Canine dysautonomia in a litter of Havanese puppies.

Canine dysautonomia is a sporadic, generally fatal disease that rarely affects groups of related animals. Four 10-week-old Havanese puppies from a litter of 5 developed clinical signs of canine dysautonomia. The 4 affected dogs were exposed to an outdoor environment, whereas the fifth littermate was not exposed to the outdoors and remained clinically healthy. Clinical signs of dysautonomia developed 10-16 days after going outside the house. An unrelated dog also developed dysautonomia after exposure to 1 of the affected Havanese littermates. All 5 dogs had morphological changes consistent with dysautonomia (widespread neuronal degeneration in autonomic ganglia, select brainstem nuclei, and ventral horn motor neurons). Differential diagnoses were excluded through negative toxicological evaluation, fecal parasite screening, negative Canine distemper virus reverse transcription polymerase chain reaction, fluorescent antibody testing, attempted virus isolation, and electron microscopy. The 5 affected dogs were in the Kansas City, Missouri area, where there is a high incidence of dysautonomia.

Evaluation of the Efficacy, Potential for Vector Transmission, and Duration of Immunity of MP-12, an Attenuated Rift Valley Fever Virus Vaccine Candidate, in Sheep.

Rift Valley fever virus (RVFV) causes serious disease in ruminants and humans in Africa. In North America, there are susceptible ruminant hosts and competent mosquito vectors, yet there are no fully licensed animal vaccines for this arthropod-borne virus, should it be introduced. Studies in sheep and cattle have found the attenuated strain of RVFV, MP-12, to be both safe and efficacious based on early testing, and a 2-year conditional license for use in U.S. livestock has been issued. The purpose of this study was to further determine the vaccine's potential to infect mosquitoes, the duration of humoral immunity to 24 months postvaccination, and the ability to prevent disease and viremia from a virulent challenge. Vaccination experiments conducted in sheep found no evidence of a potential for vector transmission to 4 North American mosquito species. Neutralizing antibodies were elicited, with titers of >1:40 still present at 24 months postvaccination. Vaccinates were protected from clinical signs and detectable viremia after challenge with virulent virus, while control sheep had fever and high-titered viremia extending for 5 days. Antibodies to three viral proteins (nucleocapsid N, the N-terminal half of glycoprotein GN, and the nonstructural protein from the short segment NSs) were also detected to 24 months using competitive enzyme-linked immunosorbent assays. This study demonstrates that the MP-12 vaccine given as a single dose in sheep generates protective immunity to a virulent challenge with antibody duration of at least 2 years, with no evidence of a risk for vector transmission.

Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus.

Chicken anemia virus (CAV) is a ubiquitous pathogen of poultry. A CAV specific TaqMan-based PCR and RT-PCR assay for real-time quantitation of viral load and relative quantitation of virus-specific transcript levels was developed. Detection of viral DNA copy number from infected MDCC-CU147 cells was determined by extrapolation from a CAV plasmid-based standard curve. Viral load increased proportionally with increasing cell number harvested, increasing from 4x10(2) copies in 250 cells with 38% virus positive cells in an indirect immunofluorescence assay to 8x10(5) copies in 250,000 cells with 64% infected cells. The estimated average viral copy number per infected cell ranged from 5 to 14. Strain-specific primers were developed to distinguish between the Cux-1 and CIA-1 strains of CAV. These primers exhibited a 3 to 4 log differential in amplification comparing homologous versus heterologous virus-primer combinations. The sensitivity of the real-time assay was found to be comparable to a nested PCR assay using DNA samples from a SPF poultry flock exposed to the SH-1 strain of CAV. The real-time PCR detected from 1.7 to 4.2 target molecules in three out of four samples that were positive by nested PCR using 50% of the DNA used in the nested PCR. Relative viral transcript levels for Cux-1 and CIA-1 infected cell cultures increased proportionally with increasing cell numbers harvested for RNA extraction. This assay will be important for both diagnosis and in understanding the complex pathogenesis of CAV infection.