Virus

The chicken parvovirus ABU strain was originally isolated from chickens in Hungary in 1984 18. For further studies, the ABU virus was subsequently purified by cesium chloride density-gradient centrifugation 16. The cesium chloride-purified ABU virus (kindly provided by J. Kisary) was used to prepare virus as inoculum for chickens, as previously described 16. Briefly, 1-day-old specific-pathogen-free (SPF) white-rock broiler chickens (n  =  10) were received from the Southeast Poultry Research Laboratory (SEPRL) flocks and inoculated orally with the cesium chloride-purified ABU virus. Seven days later, the chickens were killed and the entire intestinal portion was collected for further processing. A 10% homogenate of intestinal tissue was prepared in phosphate-buffered saline and clarified by centrifugation at 775 × g, and the supernatant was used to inoculate the chickens.

Chickens

SPF white-rock chickens were obtained from the SEPRL flocks at 3 days of age and divided into two groups of 15 birds each. Among other agents, these flocks are monitored for avian reovirus, adenovirus, infectious bursal disease virus, and chicken anemia virus 31. Chickens were housed in Horsfal isolators (Federal Designs, Inc., Comer, GA) with ad libitum access to feed and water. General care was provided as required by the Institutional Animal Care and Use Committee, as outlined in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching 8.

Experimental Infection of Chickens With ABU Parvovirus

One group of 15 birds (group A) was inoculated at 3 days of age with 0.1 ml of the chicken parvovirus ABU strain by oral routes. The other group (group B) was not inoculated and was kept as control birds. Cloacal swabs and plasma were collected at 4, 7, 10, 14, 21, 28, and 35 days postinfection (DPI) from each bird and were processed for DNA extraction using DNeasy blood and tissue kit (Qiagen, Inc., Valencia, CA), including 5 µg of carrier RNA. These DNA samples then were used in PCR amplification with a parvovirus-specific PVF1 and PVR1 primer set (see details for PCR assay below), and a total of 20 randomly selected positive amplicons were cloned and sequenced.

Clinical Samples

Enteric samples were collected from commercial turkey and chicken flocks from different regions of the United States. The field samples were received as whole guts or fecal-composite samples. A total of 138 chicken and turkey samples consisted of pooled intestines, from three birds per house, from each of a total 54 chicken and 29 turkey farms. Sixty samples were from chicken or turkey flocks less than 2 wk of age; 78 samples were collected from birds between 2 and 7 wk of age. Information on flock condition or performance was not provided for all samples. Samples were collected between 2003 and 2008. All samples were preserved at 4 C or −70 C until shipment on wet-ice (by overnight courier) to SEPRL (USDA, Athens, GA), where samples were processed within 24 hr of receipt or kept at −80 C before processing. Enteric tissues were processed for further studies as follows: approximately 1 g intestinal tissue was homogenized with a fast-prep glass bead homogenizer (Savant Instruments, Holbrook, NY) in 0.5 ml sterile 50% Dulbecco's modified Eagle's medium and 50% F12 medium (Mediatech, Herndon, VA). Samples were clarified by centrifugation for 15 min at 15,000 × g. Supernatants were used for DNA extraction using DNeasy blood and tissue kit (Qiagen), including 5 µg of carrier RNA.

Development and Evaluation of PCR Procedure

DNA was extracted from individual chicken and turkey enteric samples as described above. The complete parvovirus NS gene was amplified from extracted DNA using a 50 µl reaction mix that contained 25 µl HotStarTaq DNA polymerase (Qiagen) and 20 pmol of the previously described NS gene-specific forward and reverse primer sets NSF1/ NSMR1 and NSMF1/NSR1 in order to create overlapping segments 39. PCR was performed using a 15-min incubation at 95 C followed by 30 amplification cycles (94 C for 30 sec, 55 C for 1 min, and 68 C for 3 min). Three sets of overlapping amplicons from chicken samples, and three sets of overlapping amplicons from turkey samples representing six individual full-length NS gene sequences, were purified with a PCR purification kit (Qiagen) and eluted in 20 µl of the elution buffer (Tris-ethylenediaminetetraacetic acid) provided by the manufacturer. Five microliters of the eluted, purified PCR product were ligated to the vector TOPO-Blunt (Invitrogen, Carlsbad, CA) and used to transform E. coli TOP-10 cells (Invitrogen) according to the manufacturer's instruction. Bacteria were plated on Luria Bertani (LB) agar plates containing kanamycin at 50 µg/ml. Colonies were inoculated into LB broth (containing kanamycin) in 96-well plates and incubated at 37 C for 24 hr in a shaker incubator. DNA was prepared using a PerfectPrep Plasmid purification kit (Eppendorf North American, Westbury, NY), sequencing was performed with M13 forward and reverse primers, and the reaction products were separated on an AB-3730 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA). The final DNA consensus sequence had an average eightfold redundancy at each base position.

The sequences of the parvovirus NS gene from the six enteric samples were aligned with the previously published chicken parvovirus and turkey parvovirus NS sequences 39 using the ClustalW program 35. PCR primers were designed from regions of the aligned sequence showing high levels of nucleotide conservation and were designated as PVF1 and PVR1. Primer PVF1 (5′-TTCTAATAACGATATCACTCAAGTTTC-3′) was at position 1431–1457 nucleotide (nt) in the NS gene sequence while PVR1 (5′-TTTGCGCTTGCGGTGAAGTCTGGCTCG-3′) was at position 1965–1991 nt. To test reaction conditions, extracted DNA from ABU parvovirus-inoculated chicken enteric homogenates (ABU DNA) was used as a template for PCR. The 50-µl reaction mix contained 25 µl HotStar Taq solution (Qiagen) and 20 pmol of each of the PVF1 forward and PVR1 reverse primers. After 15 min incubation at 95 C, 30 cycles of amplification (94 C for 30 sec, 55 C for 1 min, and 68 C for 1 min) were performed. PCR products were visualized after separation in an ethidium bromide stained agarose gel.

To test the sensitivity of the assay, a 1081-base pair (bp) PCR fragment was amplified from ABU DNA using primers NSMF1 and NSR1. The amplicon containing the PVF1 and PVR1 annealing sites was cloned into a TOPO-TA vector to create a 5012-bp double-stranded DNA product. DNA was purified by a miniprep DNA purification kit (Qiagen) and used as a target to determine the number of DNA molecules the PCR assay could detect. Tenfold serial dilutions of the target DNA were used, and PCR reactions containing the PVF1 and PVR1 primers were performed as described above. Assay sensitivity was calculated based on the DNA concentration of each dilution and on length of the template DNA molecules.

To determine the specificity of the PCR assay, target DNA was extracted from the following materials as described above: uninfected primary chicken embryo fibroblast (CEF) and chicken embryo liver (CEL) cell cultures; allantoic fluid of uninfected 9-day-old embryonating chicken eggs; TOPO-TA vector plasmid and Escherichia coli; enteric homogenates of uninfected SPF chickens; and enteric homogenates containing enteric viruses (astrovirus, reovirus, rotavirus, coronavirus, adenovirus) 21.

Phylogenetic Analysis

PCR amplicons using primer sets PVF1 and PVR1 from selected parvovirus-positive field samples were used for subsequent phylogenetic analysis. A total of 38 positive PCR amplicons (using PVF1 and PVR1 primer sets) from field enteric samples were cloned and sequenced with M13 forward and reverse primers. Ten chicken and 10 turkey field parvovirus sequences were used to construct a phylogenetic tree. The amplicons were purified and ligated into the TOPO-TA cloning vector, and this was used to transform TOP-10 E. coli cells. DNA preparation and sequencing were performed as described above. Phylogenetic and molecular evolutionary analyses were conducted using MEGA Version 4.0 software package 32 ( http://www.megasoftware.net/mega4).

Development of A Conventional PCR to Detect Chicken and Turkey Parvoviruses

Based on multiple alignment data of three chicken and three turkey parvovirus NS gene sequences, two regions were found, at positions 1431–1457 nt and 1965–1991 nt within the NS gene, that showed a high level of nucleotide conservation (Fig. 1B). Using primers from those sequences, approximately 100–500 parvovirus DNA equimolecules could be detected by a conventional PCR assay (Fig. 1C). No parvovirus DNA was detected by PCR using template DNA extracted from uninfected CEF and CEL cells, allantoic fluid, vector plasmid, and E. coli and enteric homogenates containing astroviruses, reoviruses, rotaviruses, coronaviruses, or adenoviruses.

Evaluation of The PCR Assay to Detect Parvovirus in Cloacal Swab and Plasma Samples of Experimentally Infected Chickens

SPF chickens were experimentally infected with ABU parvovirus and cloacal swab samples and plasma samples were taken at various times postinfection. PCR analysis of these samples showed the presence of parvovirus as early as 4 and 7 DPI in cloacal swabs and plasma samples, respectively (Fig. 2). By 14 DPI, 100% of both swab and plasma samples became positive, and the virus persisted at a detectable level in infected birds until 28 DPI. No positive PCR amplification was observed with samples collected from uninfected SPF birds during the study.

A total of 20 randomly selected PCR amplicons, amplified from cloacal swab and plasma samples using PVF1 and PVR1 primers, were cloned and sequenced. All of those sequences were identified as parvoviruses, and amplification of nonparvovirus DNA sequences was not observed in the study.

Prevalence of Parvovirus Infections in Chicken and Turkey Flocks in The United States

A total of 138 samples from 10 different states were tested by PCR for parvovirus presence (Table 1). Of the chicken samples that were collected in 54 farms, 77% (46/60) were positive for parvovirus, while 78% (61/78) of the turkey samples that were received from 29 farms were parvovirus positive. Parvovirus infection was detected in bird samples from 8 different states including GA, SC, NC, DE, MO, AR, MN, and CA. There was no direct correlation between the age of the birds and virus presence. PCR-positive samples could be identified between 5 days and 8 wk of age in both chicken and turkey enteric samples.

Phylogenetic Studies Using Chicken and Turkey Parvovirus Ns Gene Sequences

Phylogenetic analysis revealed that the chicken and turkey parvoviruses clustered in separate groups, with the exceptions of the Ch841AR05 chicken virus that grouped with the turkey viruses and the Tu260CA05 turkey virus that grouped with the chicken parvoviruses (Fig. 3). The overall nucleotide sequence identity among chicken isolates was 95.4%–98.6%, while the turkey isolates revealed somewhat higher identities with each other, ranging within 99.3%–99.6%. There was a 90.0%–90.5% mean nucleotide sequence identity between the clustered chicken and turkey parvovirus NS gene regions.