DNA-vaccine groups and plasmid combinations.

<p>DNA-vaccine groups and plasmid combinations.</p

Characterization and evaluation of the effect of co-delivering chIFNγ with a DNA vaccination system.

<p>Plasmids expressing NDV F and chIFNγ genes were developed and characterized for their use as DNA vaccines and adjuvant, respectively. DF-1 cells were transfected with pTriEX, pTriEX-ZJ1-F and pTriEX- IFNγ. Cell culture supernatants were tested by western blotting for the presence of F protein <b>(A)</b> and chIFNγ <b>(B)</b>, respectively. Eighteen-day-old SPF ECEs were inoculated with TE buffer, pTriEX, pTriEX-ZJ1-F, or pTriEX-ZJ1-F plus pTriEX- IFNγ and boosted 2 weeks after hatched. Two weeks after booster vaccination, birds were challenged with vZJ1. Oropharyngeal <b>(C)</b> and cloacal <b>(D)</b> swab samples were collected 3 days after challenge to measure the amount of challenge virus shed into the environment. Viral titers were determined by quantitative real time reverse transcription polymerase chain reaction (qRRT-PCR). A standard was prepared with a vZJ1 virus stock of know concentration, this was included in every plate and was used to obtain viral titers expressed as EID₅₀/mL. Morbidity <b>(E)</b> and mortality <b>(F)</b> were also evaluated. Viral shedding results were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Differences in morbidity among groups were evaluated using a two-tailed Z test for comparison of sample proportions. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a <i>P<0</i>.<i>05</i>. Significant differences are denoted by different letters.</p

Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.

<p>Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.</p

In situ hybridization to identify transcription of cf76 in C. felis-infected lung tissue.

<p>A. Hematoxylin and eosin stained lung tissue demonstrating schizonts forming a parasitic thrombus within a pulmonary vessel, 20X, B. Negative sense riboprobe, hematoxylin and eosin counterstain, numerous positive cells (brown) are demonstrating intracytoplasmic presence of cf76 antigen, 20X, C. Irrelevant negative sense riboprobe, hematoxylin and eosin counterstain, 20X.</p

Primer sequences for amplification of cf76.

<p>Primer sequences for amplification of cf76.</p

Conserved gene synteny between <i>T. parva</i> p67 and <i>C. felis</i> cf76.

<p>cf76 is identified <i>in silico</i> within a highly conserved syntenic block of genes similarly to the leading vaccine candidate for <i>T. parva</i>, p67.</p

Sequence assembly of <i>Cytauxzoon felis</i> genomic and cDNA.

a<p>Expressed Sequence Tags.</p>b<p>The contig length such that using equal or longer contigs produces half the bases of the assembly.</p

Distribution of cytauxzoonosis in the United States.

<p>Distribution of cytauxzoonosis in the United States.</p

Assessment of purified cf76 and cf76 fragments by western blot.

<p>Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with anti-HIS N-terminal tag (A) and anti-HA C-terminal tag antibodies (B).</p

Assessment of feline sero-reactivity to cf76 and cf76 fragments by western blot.

<p>Purified full length cf76 (1), the N-terminal region (2), the central region (3), and the C-terminal region (4) were probed with pooled sera (1∶500) from cats surviving <i>C. felis</i> infection (A) or naive cats (B).</p