Genotyping of turkey coronavirus field isolates from various geographic locations in the Unites States based on the spike gene.

Turkey flocks have experienced turkey coronaviral enteritis sporadically in the United States since the 1990s. Twenty-four field isolates of turkey coronavirus (TCoV) from multiple states in the United States were recovered from 1994 to 2010 to determine the genetic relationships among them. The entire spike (S) gene of each TCoV isolate was amplified and sequenced. Pairwise comparisons were performed using the Clustal W program, revealing 90.0% to 98.4% sequence identity in the full-length S protein, 77.6% to 96.6% in the amino terminus of the S1 subunit (containing one hypervariable region in S1a), and 92.1% to 99.3% in the S2 subunit at the deduced amino acid sequence level. The conserved motifs, including two cleavage recognition sequences of the S protein, two heptad repeats, the transmembrane domain, and the Golgi retention signal were identified in all TCoV isolates. Phylogenetic analysis based on the full-length S gene was used to distinguish North American TCoV isolates from French TCoV isolates. Among the North American TCoV isolates, three distinct genetic groups with 100% bootstrap support were observed. North Carolina isolates formed group I, Texas isolates formed group II, and Minnesota isolates formed Group III. The S genes of 24 TCoV isolates from the United States remained conserved because they contained predominantly synonymous substitutions. The findings of the present study suggest endemic circulation of distinct TCoV genotypes in different geographic locations.

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 µg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

Production of recombinant Bartonella henselae 17-kDa protein for antibody-capture enzyme-linked immunosorbent assay.

The Bartonella henselae 17-kDa protein was expressed in a prokaryotic expression system as a histidine-tagged fusion protein and was purified. The target gene was cloned into a recombinant expression construct, pTri-17kd. The expressed protein was purified to near homogeneity by a nickel-agarose column chromatography. Protein recovery was estimated to be 2.9 mg from 100 mL of bacterial culture. The purified 17-kDa protein was recognized by serum from patients infected with B. henselae and Bartonella quintana, suggesting antigenic integrity. The sensitivity and specificity of the IgG enzyme-linked immunosorbent assay (ELISA) relative to immunofluorescent antibody assay testing were 71.1% and 93.0%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.823. These results indicate that the expressed 17-kDa protein is a suitable source of antigen for development of an antibody-capture ELISA for the detection of antibodies to B. henselae.

Comparison of 3'-end encoding regions of turkey coronavirus isolates from Indiana, North Carolina, and Minnesota with chicken infectious bronchitis coronavirus strains.

OBJECTIVE: To analyze the 3'-end structural protein-encoding region of turkey coronavirus (TCoV) isolates associated with outbreaks of acute enteritis in Indiana, North Carolina, or Minnesota. METHODS: Four isolates of TCoV were sequenced over the entire 3'-end structural protein-encoding region and compared phylogenetically along with the corresponding sequences of infectious bronchitis virus (IBV) strains. RESULTS: The sequence similarity between TCoV and IBV was lower than that among TCoV isolates or that among IBV strains. The variation of sequences between TCoV and IBV was mainly contributed by the S protein gene. The sequence similarity of S gene between TCoV and IBV was lower than that among TCoV isolates or that among IBV strains. The phylogenetic tree based on the S protein region was similar to that based on the entire 3'-end structural protein-encoding region with TCoV isolates and IBV strains grouped in two separate clusters. The phylogenetic tree based on other genes had a very different topology with TCoV isolates randomly forming groups with different IBV strains. CONCLUSIONS: These results suggested that TCoV probably shared the same origin with that of IBV and acquired sequences of S gene for turkey intestine tropism during the process of evolution in a separate environment.

Complete sequences of 3' end coding region for structural protein genes of turkey coronavirus.

Overlapping fragments of genomic RNA spanning 6963 nucleotides from 5' end of spike (S) protein gene to 3' end of nucleocapsid (N) protein gene of turkey coronavirus (TCoV) were amplified by reverse-transcription-polymerase chain reaction (RT-PCR). The primers were derived from the corresponding sequences of infectious bronchitis virus (IBV). The PCR products were cloned and sequenced and their nucleic acid structure and similarity to published sequences of other coronaviruses were analyzed. Sequencing and subsequent analysis revealed 9 open reading frames (ORFs) representing the entire S protein gene, tricistronic gene 3, membrane (M) protein gene, bicistronic gene 5, and N protein gene in the order of 5'-3'. The overall nucleic acid structures of these encoding regions of TCoV were very similar to the homologous regions of IBV. The consensus transcription-regulating sequence (TRS) of IBV, CT(T/G)AACAA, was highly conserved in TCoV genome at the levels of nucleotide sequence and location in regarding to the initiation codon of individual genes. Pair-wise comparison of gene 3, M gene, gene 5, or N gene sequences with their counterparts of IBV revealed high levels (82.1-92.0%) of similarity. Phylogenetic analysis based on the deduced amino acid sequences of S, M, or N protein demonstrated that TCoV was clustered within the same genomic lineage as the IBV strains while all the other mammalian coronaviruses were grouped into separate clusters corresponding to antigenic groups I or II. There were substantial differences of S protein sequence between TCoV and IBV with only 33.8-33.9% of similarity.

Comparison of Western immunoblotting and the C6 Lyme antibody test for laboratory detection of Lyme disease.

Three commercial Lyme disease Western immunoblotting (WB) kits and the C6 Borrelia burgdorferi (Lyme) enzyme-linked immunosorbent assay (ELISA) kit were compared using two commercially available performance panels from the Centers for Disease Control and Prevention (CDC) and Boston Biomedica (BBI). Combined, the panels consisted of 52 characterized specimens. Immunoglobulin G (IgG) sensitivity was similar for the three WB products. The BBI and Marblot WBs were more specific for IgG antibodies, while the Virablot was the most sensitive for IgM antibody. The BBI WB was 100% specific for IgM, while Marblot was 97% and Virablot was 77% specific for IgM. The C6 ELISA was found to be 100% sensitive. Four false-positive C6 results were identified in patients that had clinically and microbiologically confirmed Lyme disease but were not detected by the CDC reference methods. No one WB product showed overall superiority. The C6 ELISA shows promise as the first ELISA for Lyme disease that would not require a supplemental test such as a WB.

Serological diagnosis of human babesiosis by IgG enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies to Babesia microti antigen was developed. B. microti antigens were harvested from experimentally infected hamster blood and used as a coating antigen. The sensitivity and specificity of the IgG ELISA relative to immunofluorescent antibody assay (IFA) testing was 95.5% and 94.1%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.987. No cross-reactivity of serum samples collected from patients infected with Toxoplasma gondii, Borrelia burgdorferi, Anaplasma phagocytophilum, Bartonella quintana, Dengue virus, or West Nile virus was detected. Cross-reactivity was observed with one of 35 sera from patients infected with Bartonella henselae. These results indicate that the established ELISA methods could be utilized as an accurate measure for the clinical diagnosis of human babesiosis.

Antigenic relationship of turkey coronavirus isolates from different geographic locations in the United States.

The purpose of the present study was to examine the antigenicity of turkey coronavirus (TCV) isolates from various geographic areas with antibodies to different viruses. Seventeen isolates of TCV were recovered from intestinal samples submitted to Animal Disease Diagnostic Laboratory, Purdue University, from turkey farms located in different geographic areas. The prototype TCV Minnesota isolate (TCV-ATCC) was obtained from the American Type Culture Collection. Intestinal sections were prepared from turkey embryos infected with different TCV isolates and reacted with polyclonal or monoclonal antibodies to TCV, infectious bronchitis virus (IBV), bovine coronavirus (BCV), transmissible gastroenteritis virus (TGEV), reovirus, rotavirus, adenovirus, or enterovirus in immunofluorescent antibody staining. All 18 TCV isolates have the same antigenic reactivity pattern with the same panel of antibodies. Positive reactivity was seen with polyclonal antibodies to the TCV Indiana isolate, the TCV Virginia isolate, TCV-ATCC, and the IBV Massachusetts strain as well as monoclonal antibodies to the TCV North Carolina isolate or the membrane protein of IBV. Antibodies to BCV or TGEV were not reactive with any of the TCV isolates. Reactivity of antibodies to unrelated virus, rotavirus, reovirus, adenovirus, or enterovirus with different TCV isolates was all negative, except positive response was seen between enterovirus antibody and a TCV western North Carolina isolate, suggesting coinfection of turkeys with TCV and enterovirus in that particular case. The results indicated that the TCV isolates from these geographic locations in the U.S. shared close antigenicity and were antigenically related to IBV.

The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus.

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkey's were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2-3 fold decreased (p < 0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p < 0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p < 0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1-2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p < 0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.