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Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis
Clinical Proteomics volume 15, Article number: 44 (2018)
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Cytauxzoonosis is a disease of felids in North America caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis. Cytauxzoonosis is particularly virulent for domestic cats, but no vaccine currently exists. The parasite cannot be cultivated in vitro, presenting a significant limitation for vaccine development.
Recent sequencing of the C. felis genome has identified over 4300 putative protein-encoding genes. From this pool we constructed a protein microarray containing 673 putative C. felis proteins. This microarray was probed with sera from C. felis-infected and naïve cats to identify differentially reactive antigens which were incorporated into two expression library vaccines, one polyvalent and one monovalent. We assessed the efficacy of these vaccines to prevent of infection and/or disease in a tick-challenge model.
Probing of the protein microarray resulted in identification of 30 differentially reactive C. felis antigens that were incorporated into the two expression library vaccines. However, expression library immunization failed to prevent infection or disease in cats challenged with C. felis.
Protein microarray facilitated high-throughput identification of novel antigens, substantially increasing the pool of characterized C. felis antigens. These antigens should be considered for development of C. felis vaccines, diagnostics, and therapeutics.
Cytauxzoon felis is a tick-transmitted apicomplexan parasite that is the causative agent of cytauxzoonosis in domestic and wild felids in North and South America [1,2,3,4,5,6,7,8]. A closely related, genetically unique Cytauxzoon sp. has been identified in Europe [9,10,11,12,13], but has not been associated with classic cytauxzoonosis which is characterized by high mortality rates and massive proliferation and vascular dissemination of schizont-infected myeloid cells. Although no longer considered uniformly fatal in domestic cats, morbidity and mortality remain high for individuals presenting with acute cytauxzoonosis [14,15,16,17,18,19]. Even with treatment, which can cost thousands of dollars, mortality remains at least 40% [20]; a vaccine is not currently available. Following its initial discovery in Missouri in 1976, C. felis has since been detected in domestic cats in 19 states [6, 8, 21, 22] and in bobcats in two additional states [23, 24]. The prevalence of C. felis infections in enzootic regions of the USA has been reported at 6.2% [17], which is higher than the estimated prevalence rates of common infections like feline leukemia virus (2.26%) and feline immunodeficiency virus (4.2%, Companion Animal Parasite Council, https://capcvet.org/maps/#2017/all/felv/cat/united-states/, https://capcvet.org/maps/#2017/all/fiv/cat/united-states/). This pattern could be due to a combination of factors, including an increase in feline reservoirs, expanding range of competent tick vectors (Amblyomma americanum and Dermacentor variabilis), and increased clinician awareness and diagnosis.
Prevention of cytauxzoonosis appears to be the optimal control strategy, and currently depends either on application of prophylactic acaricides [25], or keeping cats indoors. However, in practice, these strategies are flawed. Despite recommendations to house cats indoors, an estimated 35–60 million pet cats are still allowed to roam outdoors in the United States [26, 27]. Furthermore, effective acaricide prophylaxis may be limited by lack of owner compliance or cost of acaricides, as a recent study indicated as few as 38% of cats presenting to a veterinary teaching hospital had not received any form of tick prevention [28]. We therefore propose that vaccination against C. felis would serve as a practical and cost-effective method of prevention. Because cytauxzoonosis is highly pathogenic, a vaccine that reduces morbidity and mortality, even if it failed to prevent infection, would be considered successful.
There is some evidence that domestic cats can develop a protective immune response against C. felis. Previous studies have demonstrated that cats that survive acute cytauxzoonosis are protected from clinical disease following subsequent challenge with a lethal dose of virulent C. felis [4, 29,30,31]. However, antigen discovery and vaccine development have been hindered by the inability to cultivate C. felis in vitro. To redress this we previously sequenced and annotated the C. felis genome, and undertook a search for vaccine candidates [32] via an approach known as "reverse vaccinology" [33]. The underlying premise is that each of the 4300 putative proteins that were predicted [32] could represent a potential vaccine candidate. To date, a single C. felis vaccine candidate has been identified and characterized using a single candidate gene approach [32]. Although identification of candidates in such fashion is conceptually straightforward, this approach is both labor-intensive and cost-prohibitive when considering the number of inferred protein-encoding genes present in the C. felis genome. As a more efficient strategy, we deployed a protein microarray to screen a large number of C. felis proteins for antigenicity. Protein microarray technology has been utilized as a strategy for identification and assessment of diagnostic and/or vaccine candidates for a variety of pathogens, including protozoal organisms [34,35,36,37,38,39] as well as fastidious organisms that have not been cultivated in vitro [40, 41]. Conceptually similar, an alternative, high throughput method for screening antigens as vaccine candidates in vivo is a DNA expression library immunization (ELI) approach. This entails immunization of individuals with a library of expression plasmids containing either a portion of or the entire genome of the infectious agent [42,43,44]. ELI has been used to identify protective antigens against a variety of protozoal infections in mice, including Plasmodium, Leishmania, and Trypanosoma species [42, 45,46,47,48,49].
Our goal was to develop a low-cost, fast-track approach for vaccine development that could be used for pathogens that are difficult to isolate/culture or for which there is limited funding available. The aims of this study were twofold. First, we described the utilization of a C. felis protein microarray for identification of antigens. Then we assessed the ability of these antigens to induce protection from clinical disease via ELI. Protein microarray successfully identified 30 C. felis antigens. However, ELI using these antigens failed to induce protection against disease or infection. Collectively, these data provide insight into the humoral immune response against C. felis, and help build a foundation for future vaccine development against C. felis.
Cytauxzoon felis DNA isolation, RNA isolation and cDNA synthesis
Cytauxzoon felis genomic DNA was extracted from leukoreduced blood (Purecell NEO Neonatal High Efficiency Leukocyte Reduction Filter for Red Cell Aliquots, PALL Corp., Port Washington, NY) using a commercially available kit (QIAamp DNA Blood Mini Kit Qiagen, Valencia, CA). RNA was extracted from liver tissue of a cat infected with C. felis using a commercially available kit (ZR Tissue and Insect RNA Kit, Zymo Research, Irvine, CA). Using this RNA as template, cDNA was synthesized with Taqman Reverse Transcription Reagents following the two step RT-PCR protocol according to manufacturer's instructions (Roche, Mannheim, Germany).
Identification and selection of Cytauxzoon felis genes/ORFs
Of the 4378 predicted C. felis proteins [32], we selected 864 as candidates (Additional file 1: Supplementary Data Set 1) to assess via protein microarray based on at least one of the following criteria: genomic synteny and/or sequence homology to antigenic proteins identified in related protozoal organisms; sequence homology to Plasmodium proteins that displayed heterologous reactivity against serum from cats infected with C. felis (data not shown); or the presence of putative features predicted in silico to increase antigenicity, including the presence of predicted signal peptides, transmembrane domains, and predicted antigenic sites (Signal P Server v.4.0 and TMHMM Server v.2.0 from the Center for Biological Sequence Analysis). Of the genes/ORFs encoding the 864 putative proteins, those that were 126–3804 bp in length were selected for amplification for inclusion on the protein microarray (768/864).
PCR amplification of linear acceptor vectors (pXT7 and PVAX1)
We used high throughput homologous recombination to clone target genes for expression on the protein microarray (pXT7) and for incorporation into DNA vaccines (pVAX1). Linearized pXT7 was produced and amplified using previously described methods [50] with the following modification: Roche high-fidelity enzyme mix (2.5 U) and a 1X concentration of Expand high-fidelity buffer with MgCl2 was utilized (Roche, Mannheim, Germany).
Linearized pVAX1 (Invitrogen, Carlsbad, CA) was produced using identical methods as for pXT7. Primers were designed to amplify the linear acceptor vector and incorporate a Kozak sequence upstream of the cloning site and a stop codon downstream of the coding site (Additional file 2: Supplementary Table 1). The 25 μL reaction consisted of 1 ng of linearized pVAX1, 25 pmols of each primer (Forward: 5′-TAGATCCACTAGTCCAGTGTG-3′, Reverse: 5′-CCATGGTGGCCGAGCTCGGTACCAAGC-3′), 5 pmol of dNTPs, 1.155 U of Expand high-fidelity enzyme mix, and a 1X concentration of Expand high-fidelity buffer with MgCl2 (Roche, Mannheim, Germany). Thermal cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 amplification cycles (94 °C for 20 s, 50 °C for 30 s, and 68 °C for 3.5 min) and a final extension step at 72 °C for 5 min (Techne Inc., Burlington, NJ).
PCR amplification of Cytauxzoon felis genes/ORFs for protein microarray and DNA vaccine
For genes/ORFs included on protein microarray, we designed primers that included cassettes homologous cloning sites of the linearized pXT7 (Forward cassette: 5′-ACGACAAGCATATGCTCGAG-3′, Reverse cassette: 5′-TCCGGAACATCGTATGGGTA-3′); ORFs that were larger than 3000 bp were split into smaller fragments as needed for efficient amplification. Each 50 μL reaction contained 1 μL of genomic C. felis DNA template or cDNA, 50 pmol of each primer, 10 nmol of deoxynucleoside triphosphates (dNTPs), 2.5 U of Expand high-fidelity enzyme mix, and a 1X concentration of Expand high-fidelity buffer with MgCl2 (Roche, Mannheim, Germany). Thermal cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 40–45 amplification cycles (95 °C for 30 s, 55 °C for 15 s, 50 °C for 15 s, and 68 °C for 3 min) and a final extension step at 68 °C for 10 min (Techne Inc., Burlington, NJ).
Similarly, for candidates included in the DNA vaccine, we designed primers with cassettes homologous to cloning sites of pVAX1 (Forward cassette: 5′-ACCGAGCTCGGCCACCATGG-3′, Reverse cassette: 5′-CACACTGGACTAGTGGATCTA-3′). Each 25 μL reaction contained 1 μL of genomic C. felis DNA template or cDNA (Additional file 2: Supplementary Table 1), 25 pmol of each primer, 5 nmol of dNTPs, 1.75 U of Expand high-fidelity enzyme mix, and a 1X concentration of Expand high-fidelity buffer with MgCl2 (Roche, Mannheim, Germany). Thermal cycling conditions consisted of an initial denaturation step at 94 °C for 5 min, followed by 40 amplification cycles (94 °C for 30 s, 55 °C for 15 s, 50 °C for 15 s, and 68 °C for 4 min) and a final extension step at 68 °C for 7 min (Techne Inc., Burlington, NJ).
PCR conditions were optimized for individual genes/ORFs as needed, and amplicons were confirmed by gel electrophoresis for correct size prior to proceeding to homologous recombination.
High throughput homologous recombination cloning
All successfully amplified genes/ORFs were cloned into the appropriate plasmid expression vector (pXT7 for the microarray or pVAX1 for DNA vaccine) using a high throughput PCR recombination cloning method as previously described [50]. While the majority of plasmids cloned with pXT7 for the protein microarray were isolated without colony selection as described [50], colony selection was utilized for those plasmids cloned with pVAX1 for the DNA vaccine. Minipreps of clones were confirmed to have the correct insert via PCR using ORF sequence-specific primers and/or bi-directionally sequencing using T7 and BGH reverse primers; internal sequencing primers were designed and utilized as needed to obtain complete bi-directional sequencing (MCLAB, South San Francisco, CA).
Protein microarray printing
The expression of successfully cloned genes/ORFs was carried out for 5 h via in vitro transcription-translation (IVTT) according to manufacturer's directions (RTS E. coli HY 100 kit, biotechrabbit GmbH, Germany) with addition of 0.067 mM Brij-98. A selection of ORFs that were predicted to be soluble (n = 137) were expressed in duplicate and transferred to plates without addition of Brij-98. Crude proteins were printed immediately without purification on nitrocellulose-coated glass NOVA slides (Grace Bio Labs, Bend, Oregon 97702) using an OmniGrid 100 microarray printer (GeneMachines, San Carlos, CA). Controls spotted onto chips included IVTT reactions without plasmid and purified IgG, and protein expression was confirmed by probing for polyhistidine (clone His-1, Sigma) and hemagglutinin tags (clone 3F10, Roche) included within the PXT7 vector.
Protein microarray probing
Serum was collected from 48 cats throughout the United States (20 cats PCR positive for C. felis, 20 cats PCR negative for C. felis, and 8 SPF cats that were PCR negative for C. felis). All 20 infected cats were known to have survived acute cytauxzoonosis. Four of these C. felis infected cats were known to be acutely infected and 4 were chronically infected.
Sera were diluted to 1:200 in Protein Array Blocking Buffer (GVS, Sanford, ME) with 10% (vol/vol) E. coli lysate and incubated at room temperature for 30 min. Blocking anti-E. coli antibodies in the serum samples with E. coli lysate helps reducing background reactivity against E. coli proteins from IVTT reactions. Arrays were rehydrated in Protein Array Blocking Buffer and incubated in sera overnight at 4 °C with constant agitation. Slides were then rinsed 10 mM Tris buffer (TBS, pH 8.0) containing 0.05% Tween-20 (TTBS), then incubated in biotin-conjugated, goat anti-cat immunoglobulin (anti-IgGfcc, Jackson Immuno Research, West Grove, PA) diluted 1/200 in blocking buffer. After rinsing the slides, bound antibodies were detected by incubation with streptavidin conjugated SureLightH P-3 (Columbia Biosciences, Frederick, MD). The slides were then washed three times in TTBS and three times in TBS followed by a final water wash. The slides were air dried after brief centrifugation and analyzed using a Perkin Elmer ScanArray Express HT microarray scanner (Waltham, MA).
Protein microarray statistical analysis
The protein microarrays used here do not meet the criteria for required deposition under MIAME guidelines [51], and alternatives to standardize protein microarray results are in development to insure that all information can be easily interpreted (description of minimum information about a proteomics experiment [MIAPE] can be found here [52]). Intensities were quantified using QuantArray software utilizing automatic local background subtraction for each spot. ''No DNA'' controls consisting of E. coli IVTT reactions without addition of plasmid were averaged and used to subtract background E. coli reactivity from the unmanipulated raw data. All results presented are expressed as signal intensity. As previously reported [53], the ''vsn'' package in the Bioconductor suite (http://Bioconductor.org/) in the R statistical environment (http://www.R-project.org) was used to calculate seroreactivity. In addition to the variance correction, this method calculates maximum likelihood shifting and scaling calibration parameters for different arrays, using known non-differentially expressed spots. This calibration has been shown to minimize experimental effects [54]. We used raw values for the positive and negative controls to calibrate, and then normalize, the entire data set using the vsn package. Antigens were considered to be reactive if signal intensities were greater than the average signal intensity of the "No DNA" control spots, plus 1.5-times the standard deviation. Differential analysis of the normalized signals was then performed using a Bayes-regularized t test adapted from Cyber-T for protein arrays [55,56,57,58]. Benjamini–Hochberg p value adjustments were applied to account for multiple test conditions [59]. All p values shown are Benjamini–Hochberg corrected for false discovery.
In silico analysis of antigens selected for inclusion in vaccines
A total of 33 different C. felis antigens printed on the protein microarray were chosen for inclusion two DNA vaccines (Table 1). Each candidate was assessed for amino acid sequence similarity to other Piroplasmida proteins via BLAST search (blastx) against Theileria and Babesia species genomes (PiroplasmaDB, http://piroplasmadb.org/piro/), Plasmodium species genomes (PlasmoDB, http://plasmodb.org/plasmo/), or all banked nucleotide sequences in the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Production of vaccines
Individual colonies confirmed to contain plasmids of interest were selected and grown in 150 mL LB/kanamycin (50 µg/mL) and were isolated according to manufacturer's instructions (Zyppy Plasmid Maxiprep Kit, Zymo Research, Irvine, CA) or were commercially prepared (Genewiz, South Plainfield, NJ). Multiple maxipreparations were performed as needed to attain the desired final amount of plasmid. Plasmids were further concentrated by ethanol precipitation as necessary and were resuspended in endotoxin-free tissue culture grade distilled water (Thermo Fisher Scientific, Waltham, MA).
Two different vaccines were developed: "CF-Library" and "CF-1." CF-Library consisted of 32 candidates, while CF-1 consisted of both the carboxy-terminal region and full-length C. felis cf76 (Additional file 2: Supplementary Table 1) [32]. Each vaccine dosage contained a total of 1 mg of DNA. CF-1 contained 500 μg each of C-terminal region cf76 and full-length cf76, while CF-Library contained 31.25 μg of each of the 32 plasmids.
Eighteen, 11–18 month old, intact female purpose bred cats that were PCR negative for C. felis were obtained from a commercial supplier (Liberty Research, Inc., USA). Cats were cared for according to the principles outlined in the National Institutes of Health Guide for the Care Use of Laboratory Animals and were housed in AAALAC accredited facilities within sealed, climate-controlled isolation rooms with 12 h light/dark cycles. All animal use was approved by the University of Missouri Animal Use and Care Committee (protocol number 7909).
Vaccine study design
Cats were randomly assigned to test and control groups, which are summarized in Additional file 3: Supplementary Fig. 1. Seven cats received vaccines: three cats (331, 623, and 638) received CF-1 and four cats (77, 308, 339, and 835) received CF-Library. Investigators were blinded to whether vaccinated cats received CF-Library or CF-1. Eleven cats did not receive vaccines: eight of these were infected with C. felis (positive controls for infection) and three were infested with C. felis-naïve ticks (negative controls for infection). Unvaccinated cats were also simultaneously enrolled in separate studies testing the efficacy of novel chemotherapeutics against cytauxzoonosis and the immune response to tick infestation and/or C. felis infection.
The schedule of vaccination and infection is summarized in Additional file 4: Supplementary Fig. 2. Cats within both vaccine test groups received three intramuscular (IM) injections. For the first two vaccinations (Day 6 and Day 30), cats received 1 mg of DNA (0.7 mL volume) via needle and syringe in the left (Day 6) or right (Day 30) cranial thigh (quadriceps femoris muscle). The third and final vaccination (1 mg of DNA in 0.25 mL) was administered on Day 50 in the caudal medial right thigh via VET JET transdermal vaccination system (Merial, Inc., Athens, Georgia). When using the VET JET device, cats were sedated with dexdomitor (15–20 ug/kg) and acepromazine (0.1 mg/kg) delivered intravascularly (IV).
Challenge model
Cats were infected with C. felis 59 days after initial vaccination (Additional file 4: Supplementary Fig. 2) using tick transmission as previously described [25]. Uninfected cats were identically infested with C. felis-naïve ticks. For tick infestation, cats were anesthetized as described for transdermal vaccination above. Ticks were removed from cats 10–13 days post infestation, and the number of free and attached ticks as well as subjective engorgement of ticks were recorded for each cat (Additional file 5: Supplementary Table 2).
Clinical evaluation
Once infected cats were inspected at least four times a day. Physical exams were performed daily, and attitude, heart rate, respiratory rate, and body temperature (measured rectally or by subcutaneous temperature chip (IPTT-3000, Bio Medic Data Systems, Seaford, DE)) were recorded 2–4 times daily.
Starting at 12 days post-infection and finishing at the resolution of clinical symptoms or death, blood was collected from each cat via the jugular vein or a subcutaneous venous access port (VAP; CompanionPort CP4, Access Technologies, Skokie, IL). Blood was collected into EDTA for complete blood count (CBC; Sysmex XT2000i V Automated Hematology Analyzer) and into red top tubes for serum biochemical profile (Olympus 400AUe Chemistry Analyzer). Frequency of hematologic and biochemical testing performed for each individual cat was dictated by severity of illness and discretion of attending veterinarian but was generally performed every-other-day, and peaks and/or nadirs of clinicopathologic parameters were compared.
Therapeutic intervention
Supportive care and/or antiprotozoal therapy was initiated on a case by case basis at the discretion of attending veterinarian based on assigned treatment group (unvaccinated infected cats) or at the discretion of attending veterinarian if treatment was unassigned (vaccinated cats). Specific supportive therapies administered varied between cats and were tailored to the clinical needs of the individual (Additional file 6: Supplementary Table 3). Three different antiprotozoal treatment regimens were utilized in this study: two were experimental therapies that were administered to unvaccinated infected cats as a part of a separate study (Additional file 3: Supplementary Fig. 1). Experimental therapies included either Coartem [20 mg artemether + 120 mg lumefantrine/tablet (Novartis), 2 tablets BID for 3 days] or a combination of 4 oral antibiotics, referred to through the study as "4X Abx" (pradofloxacin 7.5 mg/kg q24 h, doxycycline 10 mg/kg q12 h, metronidazole 25 mg/kg q12 h, clindamycin 10 mg/kg q12 h until clinical improvement). For any cat that developed advanced cytauxzoonosis despite vaccination or experimental treatments, atovaquone (Mepron, GlaxoSmithKline) and azithromycin was administered as previous described [20].
Assessment of infection, disease, and survival
Infection was confirmed by PCR amplification of parasite DNA (ITS-1 and/or 18S) as previously described [60,61,62] and if possible by identification of parasites via microscopic evaluation of blood smears. Cytauxzoonosis was defined as cats being febrile (body temperature ≥ 102.5 °F) and having at least one of the following three biochemical/hematological abnormalities: hyperbilirubinemia (> 0.3 mg/dL), neutropenia (< 2.5 × 103 segmented neutrophils/µL), or thrombocytopenia (< 300 × 103 platelets/µL). Survival rates of groups were compared using the Freeman Halton extension [63] of the Fisher exact probability test with significance set at p ≤ 0.05 (VassarStats, Poughkeepsie, NY).
Assessment of humoral responses post vaccination and challenge
After the completion of the clinical trial portion of the vaccine pilot study, C. felis protein microarrays were probed as described above with sera obtained from cats from each test group. Sera were collected from vaccinated cats at 5–7 different time points throughout the study and were frozen at − 80 °C until protein microarray analysis (Additional file 4: Supplementary Fig. 2).
Construction and quality assessment of Cytauxzoon felis protein microarray
Of the 864 C. felis genes/ORFs initially selected for analysis, 96 ORFs were excluded from further study due to small size and predicted minimal antigenicity and 48 ORFs were unable to be PCR amplified, leaving 720 genes/ORFs amplified for cloning. Of these, 15 were unable to be cloned, and 32 had an incorrectly-sized insert in the pXT7 plasmid, leaving a total of 673 proteins for printing on the protein microarray (93.5% cloning efficiency). Of these proteins, 633 proteins (94.07%) were efficiently expressed as determined by dual positive signals against the amino-terminal HIS and carboxy-terminal hemagglutinin (HA) tags.
Identification of C. felis antigens and selection of vaccine candidates
The antibody response profile to C. felis proteins in infected, uninfected, and SPF domestic cats is shown as a heatmap in Fig. 1. Of the total 673 genes/ORFs probed, a total of 38 (5.6%) were found to be seroreactive to feline serum. Of these, 30 were differentially reactive to serum from C. felis infected cats (p < 0.05) and 8 were cross-reactive to both infected and uninfected feline serum (p > 0.05). The number of infected and uninfected cats reacting to differentially reactive and cross-reactive proteins is summarized in Table 2.
Protein features of the 30 differentially reactive proteins include 21 proteins with a predicted signal peptide and 21 proteins with at least one and up to 11 predicted transmembrane domains (Table 1). All 30 proteins were predicted to have at least two and up to 52 antigenic sites (Table 1). Of these 30 proteins, four had been selected for placement on the microarray due to known orthology with antigenic proteins from related organisms (Table 1). One protein was selected due to homology to a conserved Plasmodium protein that previously showed reactivity against serum from C. felis infected cats (data not shown, Table 1). The remaining 25 proteins were selected for placement on the protein microarray primarily due to in silico predicted antigenicity, but on further investigation at least 15 of these proteins showed some degree of orthology to proteins from related organisms (Table 1). Ultimately, all 30 of the differentially reactive proteins were selected for incorporation into CF-Library (Table 1).
One cross-reactive protein was also selected for inclusion into CF-Library (Elongation Factor-1 orthologue). This protein was nearly classified as being differentially reactive (p = 0.052, Fig. 1), and was differentially reactive on a heterologous microarray screening sera from C. felis infected cats against Plasmodium falciparum antigens (Candidate 31, Table 1).
Of the infected feline serum samples, four were known to be collected while the cat was suffering from acute cytauxzoonosis, while four were known to be collected > 1 year after resolution of disease but while the cat remained persistently parasitemic. The antibody response profile to C. felis proteins in these samples were compared to assess for kinetic changes in serologic profile against C. felis (Additional file 7: Supplementary Fig. 3). A total of 52 proteins were found to be reactive (signal intensity > 1.5X No DNA standard deviation) to sera from either acute or chronically infected cats. No significant difference was seen (p > 0.05) for any proteins when comparing reactivity between serum of chronically or acutely infected cats. The majority of the proteins (n = 50) had a higher average reactivity against sera from chronically infected cats. However, two proteins had a higher average reactivity against sera from acutely infected cats (Additional file 7: Supplementary Fig. 3). These two proteins were also selected for inclusion in CF-Library (Candidates 32–33, Table 1).
Vaccination and challenge
Of the 33 C. felis genes/ORFs selected for ELI, 32 were successfully cloned into the expression plasmid pVAX1 (97% cloning success rate; Table 1). Both developed vaccines (CF-Library and CF-1) were delivered successfully to cats (no significant volume loss during administration), with no adverse clinical effects observed post-vaccination (Additional file 4: Supplementary Fig. 2).
All cats infested with C. felis positive ticks met the criteria for both infection and cytauxzoonosis (Table 3), with the majority (n = 13/15 infected cats) exhibiting at least two of the three biochemical/hematological abnormalities that were considered disease-defining. No parasites were detected for cats infested with C. felis-naïve ticks and those cats did not develop cytauxzoonosis.
With the exception of one cat vaccinated with CF-Library, all infected cats received supportive care and antiprotozoal therapy (Table 3, Additional file 6: Supplementary Table 3). None of the uninfected cats became ill or required treatment (Table 3).
Survival of individual cats is summarized in Fig. 2 and Table 3. Survival rate of the infected cats vaccinated against CF-Library was 100% (4 out of 4 cats survived). Survival rate of the infected cats vaccinated against CF-1 was 33% (1 out of 3 cats survived). Survival rate of the unvaccinated infected cats was 75% (6 out of 8 cats survived). Survival rate of uninfected cats was 100% (3 out of 3 cats survived). However, there was no significant difference between survival rates of groups (p > 0.05).
Retrospective serological profiles of individual cats in response to vaccination and challenge
The serological responses of individual cats to vaccination varied (Additional file 8: Supplementary Fig. 4). Five cats (all cats vaccinated with CF-Library, one cat vaccinated with CF-1) developed a positive response to at least one antigen that they were vaccinated against prior to infection with C. felis (Table 4, Additional file 8: Supplementary Fig. 4). Two cats (two cats vaccinated with CF-1) failed to develop a positive response to any antigens vaccinated against prior to infection with C. felis, and in fact, these two cats only developed positive responses to antigens that they were not vaccinated against, including Candidates 4, 15, and 31 (Table 4, Additional file 8: Supplementary Fig. 4).
Additionally, all seven vaccinated cats developed a positive response to Candidate 31 (Elongation factor-1) regardless of what they were vaccinated against, making it unclear whether the increase in seroreactivity against this candidate was C. felis-specific or not. When disregarding the positive responses against Candidate 31, only two cats developed positive responses against any other antigens they were vaccinated against. These included a positive response against either partial length cf76 (Cat 623, CF-1) or both partial and full length cf76 (Cat 77, CF-Library).
This study describes the construction and probing of a Cytauxzoon felis protein microarray for identification of antigenic proteins, the subsequent incorporation of those antigens into two expression library vaccines, and the evaluation of those vaccines to prevent clinical disease in cats challenged with C. felis infection. Using these methods, we were able to identify a pool of 30 C. felis antigens using the protein microarray, but were not able protect cats from developing cytauxzoonosis through ELI.
This study further validates protein microarray as a valuable tool for high throughput antigen discovery for organisms that are unable to be cultured in vitro. Prior to this study, only a single antigenic protein (cf76), which was identified by genome synteny with related organisms, had been characterized from a genome containing over 4300 putative proteins [32]. By use of the protein microarray, we were able to rapidly discover 30 new C. felis antigens (Fig. 1).
In this study only a portion (673/4378 = 15.4%) of the putative protein-encoding genes in the C. felis genome were analyzed. The percentage of differentially seroreactive proteins, 4.5% (30/673), was similar to percentages identified in other pathogens [34, 40, 64,65,66,67]. The pool of 673 proteins was chosen using methods to maximize the likelihood of identifying antigenic proteins. Studies evaluating the antigenicity of the remaining 3705 putative C. felis proteins should be the focus of future studies.
A limitation to this approach is that antigens identified by protein microarray are restricted to those that elicit a humoral immune response. Protection against C. felis is likely to require both a cell-mediated and humoral immune response, as the parasite resides intracellularly in the feline host. Unfortunately, high throughput methods for the identification of antigens that stimulate T-cells have only recently emerged, and are not cost-effective or widely available, particularly for use in a feline system [35, 68,69,70]. It has been suggested that proteins that induce a cellular immune response are also likely to be recognized by serum antibodies [36]. Therefore the antigens identified in this study should also be assessed for T-cell reactivity.
Unfortunately, ELI using antigens identified on the protein microarray in this study did not prevent C. felis infection or clinical disease (cytauxzoonosis). There are a number of possible explanations why ELI failed in this study. First, it is possible that failure occurred in any number of the steps required for a DNA vaccine to effectively confer immunity. This could include plasmid delivery into cells, expression of the antigens by the host cell, presentation of peptides on MHC molecules, and adequate stimulation of both innate and adaptive immune responses [42, 71]. Alternatively, vaccination could be ineffective due to inadequacies in dose, delivery route, vector, composition (lack of adjuvant), or vaccination schedule. Some of these deficiencies could be identified and overcome by assessing efficacy and immunogenicity of ELI (e.g. muscle biopsy to confirm plasmid expression, identifying uniform seroconversion) prior to challenge.
Second, it is possible that the antigens included in the vaccines were unable to induce a protective immune response against C. felis. First, recognition of a protein by antibodies does not definitively indicate a protective B-cell response [35, 36]. Furthermore, as stated previously, even if a B-cell response is generated, humoral immunity alone may not induce protection from disease. Additionally, antibodies against some of these proteins (e.g. Candidate 31, or elongation factor-1) may not represent a C. felis-specific immune response. Instead, it may represent production of cross-reactive antibodies against other pathogens (e.g. Coccidia), as it has been previously shown that serological cross-reactivity occurs between Coccidia and hemoprotozoan parasites [72, 73]. The increase in seroreactivity against these proteins (Additional file 8: Supplementary Fig. 4) could represent non-specific increases secondary to vaccination, which has previously been reported for other feline protozoan parasites [74].
We were attempting to develop a low-cost, fast-track approach for the development of a vaccine against a highly virulent feline pathogen that has never been isolated/cultured in vitro. Additional steps that should be taken in future studies include optimization and verification of the vaccination protocol prior to challenge, including verification of humoral immune responses by enzyme-linked immunosorbent assays or Western blotting.
One individual vaccinated with CF-Library (Cat 77) developed a comprehensive, albeit weak, serological response to vaccination, including a positive response to full length and the carboxy-terminal of cf76 (Additional file 8: Supplementary Fig. 4). Seven days post infection this cat's antibody titers against all candidates decreased, suggesting that these antibodies may have been depleted during early infection (Additional file 8: Supplementary Fig. 4). Interestingly, this cat was the only individual in the study that only required minimal supportive care and no antiprotozoal therapy (Table 3). It is unclear if this was due to partial protection afforded by vaccination or due to this individual cat's immune response.
In conclusion, we identified 30 new C. felis antigens via protein microarray. These antigens could be considered for use in development of diagnostic tests or therapeutic interventions against C. felis. Bedside antigen detection tests are commonly used in veterinary medicine for rapid diagnosis of virulent disease [75], and monoclonal antibodies are emerging as a therapeutic tool against diseases afflicting veterinary patients [76]. Additionally, these antigens could still be considered for use in a vaccine against C. felis using a different vaccine platform.
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Authors' contributions
MES, HM, JLT, MKS, HO, ES, DB, AV, CH, RN, LL, AT, DD, JET, ML, MR, PF, LC, and AB conceived of the study, participated in its design, and drafted, read, and approved the final manuscript. MES, HM, JLT, ES, DB, and AB performed genome analysis and selection of putative vaccine candidates. MES, HM, JLT, AV, CH, RN, and LL performed vaccine candidate cloning, and HM manufactured the DNA vaccines. MES, JLT, MKS, HO, JET, MR, and LC performed the sample collection from and caretaking of cats. CH, RN, LL, and PF performed the proteomic and bioinformatics analysis of the microarray data. All authors read and approved the final manuscript.
We would like to acknowledge the clinical expertise of Talli Hogen in the care of cats, the clinical pathology aid from Erin Burton in retrieving laboratory data, and the technical service of all veterinary technicians and students at the University of Missouri who aided in care of cats.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All pertinent data is included in this manuscript or as figures, tables, or additional files. Any additional datasets analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
All authors consent to the publication of this manuscript.
Ethics approval and consent to participate
Not applicable.
This research was funded by Morris Animal Foundation (microarray development and screening) and a charitable organization which wishes to remain anonymous.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Additional files
Additional file 1: Supplementary Data Set 1
Sequences of putative Cytauxzoon felis ORFs/genes selected for printing on protein microarray.
Additional file 2: Supplementary Table
1. PCR and cloning information for vaccine candidates.
Additional file 3: Supplementary Fig.
1. Cytauxzoon felis DNA vaccination pilot study design. A total of 18 cats were divided into four groups assessed in this study: three cats vaccinated with CF-1 prior to infection (red box), four cats vaccinated with CF-Library prior to infection (blue box), eight cats that were not vaccinated prior to infection (green box), and three cats that were neither vaccinated nor infected (gray box). The eight unvaccinated, infected cats were involved in a separate study testing the efficacy of novel chemotherapeutics against cytauxzoonosis. If it became evident that vaccines or chemotherapeutics were not halting the progression of disease, cats were additionally given atovaquone and azithromycin to attempt to prevent death; these cats are noted accordingly. A&A = atovaquone and azithromycin, 4X ABX = pradofloxacin, doxycycline, clindamycin, and metronidazole.
Additional file 4: Supplementary Fig.
2. Timeline of vaccination, infection, and sample collection for vaccinated cats.
Additional file 5: Supplementary Table
2. Additional clinical observations and information for individual cats.
Additional file 6: Supplementary Table
3. Summary of supportive care administered to individual cats. Cats vaccinated with CF-Library received less overall supportive care.
Additional file 7: Supplementary Fig.
3. Serological responses to C. felis antigens are increased during chronic infection. Serologic reactivity of antigens are depicted as a heatmap in (A). Antigens are listed in rows while grouping of individuals are denoted in columns. Average signal intensity of individual antigens against serum from acute and chronic groups are depicted in (B). The majority of antigens that had a higher average reactivity against serum from chronically infected cats (n = 50), but two antigens were more reactive against serum from acutely infected cats (n = 2, denoted with yellow boxes). These two antigens were incorporated into the CF-Library vaccine as Candidates 32-33.
Additional file 8: Supplementary Fig.
4. Serological profiles of individual vaccinated cats to candidates throughout study. Arrays containing the 32 vaccine candidates included in CF-Library and in CF-1 were probed with sera samples from individual cats. Heat map shows normalized signal intensity with red strongest, white weakest, and gray intermediate. Rows denote 32 different candidates included in vaccines listed in descending order of reactivity; candidates 19 and 3 were included in CF-1, while all listed candidates were included in CF-Library. Results are organized by individual cats (identified by number at top), and survival status of each cat is indicated as "A" (alive) or "D" (dead). Individual columns within each cat's array represent serum samples collected at different time points through study (labeled by the day in the study the sample was collected; refer to Supplemental Fig. 2 for timeline). There was no correlation between immunization protocol, individual reactivity, and survival for most cats, with the exception of Cat 77, who had widespread yet weak reactivity against all candidates in the CF-Library vaccine prior to infection and subsequently had milder disease and did not require supportive care or antiprotozoal therapy.
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Cite this article
Schreeg, M.E., Marr, H.S., Tarigo, J.L. et al. Identification of Cytauxzoon felis antigens via protein microarray and assessment of expression library immunization against cytauxzoonosis. Clin Proteom 15, 44 (2018). https://doi.org/10.1186/s12014-018-9218-9
- Cytauxzoonosis
- Cytauxzoon felis
- Expression library immunization
- Protein microarray
- Piroplasmid
- Vaccine


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After all the incredible entry's last year with a huge number of people entering, we are busy organising the Young Designers Award for the seventh time. This year's brief is based upon a new train carriage to attract more people to use the Island Train Line.
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Best Disney Books 2020: Coffee Table, Walt Disney World, Disneyland
There is an incredibly wide selection of great Disney books available on every aspect of the The Walt Disney Company. This article highlights my favorite Disney books that feature Disneyland, Walt Disney World, Disney Studios, and general Disney Company. Over time I will continue to update this page, but for now I present to you my favorite Disney books, plus new books that were released this year.
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The books are separated into categories based on the style. The categories include: historical reading, coffee table books, and in the park reading.
Historical Disney Book Reading
Disney's Land is a new addition to this list. At this point, based on all the books on Disneyland and Walt Disney I have read, it doesn't feel like there should be any new book on Disneyland I should enjoy. Somehow, author Richard Snow shares new information from a different perspective while capturing the essence of Disney in a whole new way. I would recommend this book for the casual fan and for the person who has read everything.
The Disneyland Story by Sam Gennawey is the closest thing that Disneyland has to an historical textbook. This book is constructed as the biography of Disneyland. Not only does this book provide hundreds of quotes and stories, but it provides a thorough bibliography for each chapter that provides much more thorough reading. When you are ready to go on a deep dive into the history of Disneyland, this is the book for you.
The Unofficial Disneyland Encyclopedia contains references for almost everything, every person, and place in Disneyland past and present. With explanations for items ranging from the now defunct Crocodile Mercantile store to the area previously known as Magnolia Park. Each time that you open up the book, some new tidbit will jump off the page.
The Unauthorized Story of Walt Disney's Haunted Mansion walks readers through the twisted story leading up to the creation of Disneyland's Haunted Mansion. This fascinating story could be the most unique back story to any Disneyland attraction because Walt Disney passed away and left no heir apparent for who would be the leader making the final decisions on the project. Each scene of the attraction is highlighted and dissected piece by piece. A fun read.
Before Ever After: The Lost Lectures of Walt Disney's Animation Studio consists of the manuscripts of the lectures given to the Disney Animators as the studio was transitioning from creating short films (Silly Symphonies) to their first feature film (Snow White). The book was partly compiled by legendary Disney producer Don Hahn. You can hear more about the book and Don's work at Disney in our podcast interview.
Eat Like Walt: The Wonderful World of Disney Food traces the food history that is intertwined with Walt Disney and Disneyland. The author uses an exploration of the food and restaurants in each of Disneyland's original six lands to share stories of the food of Walt Disney. The book not only dives into the food, but gives a great take on the personal life of Walt Disney the man. I flew through the stories in this book and now treasure it as a great Disney coffee table addition. This really is a must have!
Mouse Tales: A Behind-The-Ears Look at Disneyland was the first popular non-Disney approved book to appear on the market. The book is filled with stories collected from ex-Cast Member interviews. This book is lighthearted and really dishes on the past of the Disney company. Watch out if you don't want to have any of the secrets of the park divulged. This book reveals the early not-so-politically correct backstage happenings in Disneyland.
Ink & Paint: The Women of Walt Disney's Animation is a MASSIVE book covering the wide variety of roles that women played in developing early Disney Animation. The book tells incredible stories from strong females that helped to revolutionize animation film making. Through extensive interviews and behind the scenes photos, this book is equal parts text and photos. I can't decide whether to classify it as a Disney coffee table book because of its size or as a historical piece because of the incredible information offered up.
Incredible Stories Direct From Disney Cast Members
In Service to the Mouse: My Unexpected Journey to Becoming Disneyland's First President is my top recommendation for anyone who is even slightly interested in Disney history or business. This book written by Jack Lindquist, the first president of Disneyland, offers a behind the scenes look at all of the fun he had while working for the company. The book is a light read–separated into chapters by story. If you are anything like me, you will laugh out loud and finish the book the same day you pick it up.
Dream It! Do It!: My Half-Century Creating Disney's Magic Kingdoms is the memoir of Disney Legend Marty Sklar, who served as head of Walt Disney Imagineering for quite awhile. The book is written in a similar style to In Service to the Mouse, however this book focuses on the history and facts while In Service highlights the emotional situations.
The Ride of a Lifetime by Former Disney CEO Bob Iger is a fantastic read that follows his 15 year reign as the Chief Executive Officer of The Walt Disney Company. More than just a memoir, the book imparts business wisdom on how he was able to negotiate the biggest acquisitions in entertainment acquiring Pixar, Star Wars, Marvel, and 20th Century Fox. The book is surprisingly candid while easy to read. A must read for the modern Disney fan!
Windows on Main Street: 35 Years of Creating Magic at Disneyland Park is the memoir of Van Arsdale France, the man who created the world class concept of "Cast Member" training and the Disney University program. The book not only provides incredible lessons learned from his work creating the Disney training programs, but also stories ranging from the construction of Disneyland to the tough managerial style of Walt Disney. This book has been out of print of years, and was only just republished. This masterpiece is a vital addition to your Disney library.
Magic Journey: My Fantastical Walt Disney Imagineering Career is an autobiographical look back at a forty year career designing Disney's theme parks by Imagineer Kevin Rafferty. The book follows Kevin's journey from dish room attendant at Disneyland to his current position as a creative executive designing some of Disney's most impressive attractions. He worked with Jim Henson on the development of MuppetVision 3D, created the concept, story, and name for The Twilight Zone Tower of Terror, led the creation of Cars Land, and led the team responsible for Toy Story Mania. There are lots of fun stories in here and examples of Kevin's great writing.
Books About Walt Disney World Fiction
The Kingdom Keepers series are perfect books for anyone eight and up who wants a fiction book. This is a Young Adult series written by Ridley Pearson that tells the story of what happens at the Disney theme parks when the guests go home.
Disney Coffee Table Books
Walt Disney Imagineering: A Behind the Dreams Look at Making Magic Real is perhaps my favorite book in my entire Disney library. The book walks the reader through the creative process that the Disney Imagineers use while creating each new attraction, restaurant, hotel, or entire theme park. The book is filled with glossy photos and insightful information.
Walt Disney Imagineering: A Behind the Dreams Look at Making MORE Magic Real was written as a follow up to the Imagineering book I mentioned above. This book reveals even more information and updated stories based on the projects developed since the release of the first book. Once again, this is one of my favorite books in my library.
The Posters of the Disney Parks provides a unique look into the changing process of creating the masterful Disney parks posters. Also inside is a beautiful gallery highlighting the most unique Disney parks posters from around the world. This book is a great piece to leave out on the coffee table for people to flick through.
Disney Movie Posters: From Steamboat Willie to Inside Out was produced as a follow up to the successful Disney Parks Posters book explained above. The book contains every Disney movie poster from "Snow White" all the way up to "Inside Out." The book is a fun visual addition to your collection, but if I had to choose between this book and the Disney Parks Poster book, I would choose the Disney Parks book.
Designing Disney by John Hench was recently described by Disney Imagineer Kim Irvine as the book you need to read if you have any interest in Disney history, working for the company, or design. The book highlights Imagineer John Hench's life of work and advice.
The Haunted Mansion: Imagineering a Disney Classic was recently updated to highlight new changes and stories from the Haunted Mansion attraction. This book traces the creation of the attraction through stories and photos from the first conception of a haunted house in Disneyland through the completion of the Haunted Mansion in Disneyland and of the other Disney Parks around the world.
Pirates of the Caribbean: From the Magic Kingdom to the Movies is written in a similar style to the Haunted Mansion book mentioned above. The book follows the creation of the attraction through it's inspiration of the block buster Pirates of the Caribbean films. These glossy books tell the story through rare photos and stories and are great additions to your library.
Decorative Art of Victoria's Era is the original book from which Walt Disney Imagineer's drew inspiration to create the Haunted Mansion. The original concept drawing for the mansion was traced straight from pages of this book. While it was published in 1950, the price is still relatively low considering the mark that it had on Disney history.
The Art of Disneyland is one of my favorite Disneyland coffee table books as it shares incredible concept art from the over sixty years of attraction development at Disneyland. The book is now out of print and goes for crazy prices on Amazon, but is worth keeping an eye on just in case the price drops. I hope Disney circles back to this title with an update in the coming years.
Books to Pass the Time in Disneyland or Walt Disney World
These books are filled with information and written in a way that is perfect to read at home or to take with you into Disneyland. These books help to enhance the magic in the parks or bring the magic home with you.
Steven Barret has written Hidden Mickey guides for each Disney theme park around the world: Disneyland, Walt Disney World, Disney Cruise Line. These books identify the hidden Mickeys that Imagineers often incorporate into the various new attractions. Bringing this book along on your vacation turns your trip into a special scavenger hunt.
Seen, Unseen Disneyland is a photo guide to the unique design elements of Disneyland. This book will surely reveal something new to you during your next visit to Disneyland. Filled with fun photos and details from Disneyland park, this is a great way to visualize hidden details from Disneyland at home.
Lots to Do In Line, a sort of multiple choice test for the small details hidden in the queues for attractions around you, is a nice way to pass the time spent waiting in line. The questions range from recounting specific details on a sign to locating items on a scavenger hunt list. The book is great for families with young children. There are versions for both Disneyland and Walt Disney World.
The Imagineering Field Guide series is a fun pocket sized book that provides insightful tidbits of information about the history and surroundings in Disneyland and Walt Disney World. The book series includes rare concept art, interesting facts and figures, glossy photos, and incredible stories. Click here to see all of the Imagineering Field guides created for each park.
Don't Forget Disneyland Secrets!
I would be remiss if I didn't mention my new book, Disneyland Secrets: A Grand Tour of Disneyland's Hidden Details. The book is filled to the brim with secrets, each developed into a brief story that illuminates a forgotten moment in Disney history, sheds light on a neglected area of the park, or reveals something new about an iconic attraction, such as:
- Why is the address of Disneyland 1313 Harbor Boulevard?
- What's up with the Jewish menorah on Main Street?
- Why is there a fake book called "Walt & You" at City Hall?
- Where can you find Sherlock Holmes at Disneyland?
- Is there really a pet cemetery at the Haunted Mansion?
Shhh! I don't want to give it away. The answers are secret, until you read this book.
The book is now available on Amazon in both print and Kindle editions.
What are your top Disney books? COMMENT below.
These are all great Disney books to add to your collection. I also wanted to recommend some of the best stuff to watch on Disney Plus.


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Featured Articles
May Editorial – Time to Renew Our Covenant
"During those heady days in the autumn of 1979, who could have anticipated what lay ahead for the Irish Church?"
'Vocation is Today; the Christian Mission is Now'
Father Willie Purcell, National Vocations Coordinator, reflects on Pope Francis' Message for Vocations Sunday, 22 April 2018.
World Meeting of Families Icon Prayer Service
As we prepare for the WMOF in Dublin in August this year, we offer this prayer service built around the WMOF Holy Family Icon.
Muire na nGael
On 1 February, we celebrate the Feast of Saint Brigid, Abbess and Secondary Patron of Ireland. Saint Brigid is renowned for her hospitality, almsgiving and care of the sick. From the archives, Mgr Raymond Murray gives us the history of the saint affectionately known as 'Muire na nGael'. Click here to download a pdf of the
Unity is a God-Entrusted Life – Reverend Roy Cooper
A Reflection on John 17:20-26 for Christian Unity Week Be good. Take care of yourself. Have fun. Work hard. Make good decisions. Learn a lot. Be careful. Call if you need something. Remember, we love you. Those are the kind of things we say when we are leaving a son or daughter at University for
A Meditation on Luke's Account of the Birth of Christ
From our Christmas double issue, author, educator and lay missionary Frances Hogan writes on the Holy Family, and the Birth of Christ. Read the full article below, or click here to print of download a PDF of the article. Christmas! We look forward to gifts, the family gathering and all the joys of the day
Editorial – Our Annual Humanist Celebration
Far from exiting the stage of history so that humans can take their true place, it is God's entrance into history that has fully revealed to us the truth about ourselves. The conviction that God took human form is the foundation of the very highest humanism, and by the same token, Christmas is our great, annual humanist celebration.
Luther, God and Church Renewal
31 October 2017 marks 500 years since Martin Luther posted his 95 Theses on a church door in Wittenberg, Germany – the start of the Reformation movement. From our November issue, Bishop Brendan Leahy, Bishop of Limerick, writes on Luther, God and Church Renewal. Click here to download or print a pdf of the article. Luther, God and Church
World Mission Sunday: Origins and Meaning
In our latest issue of Intercom we celebrate the Mission Month of October. Fr Maurice Hogan SSC, National Director of World Missions Ireland wrote to us on the Origins and Meaning of World Mission Sunday. Until relatively recent times, the proclamation of the Gospel to non-Christians was regarded as the exclusive work of missionary societies
Sitting Where They Sit: The Irish Chaplaincy at 60
This year the Irish Chaplaincy in Britain celebrates its 60th birthday. Set up by the Irish bishops as the Irish Emigrant Chaplaincy in a very different social, political and cultural climate, it continues today to provide an outreach service to some of the most vulnerable Irish people in Britain. Since 1957, successive teams of priests,


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2020-21 Registration
Registration is now open for the 2020-21 school year. Go to "Registration" page for instructions, or to "Schedule" or "Class List" to add classes to your cart.
Life Science
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|Grade Level:
|7th - 8th
|11:00 am-12:30 pm
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|Minimum: 5, Maximum: 8
|Bonnie Netherland
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|$50.00 Supply/Lab Fee due July 1 (Non-refundable after September 1)
Life Science Student Text, BJU Press
3 Ring loose-leaf binder with 5 dividers. Set of colored markers or pencils in pouch.
|Class Description:
I will emphasize the scientific method, good lab procedures, and organizational skills (including note-taking and keeping an organized notebook). In class we will do approximately half lecture/discussion and half experiments, investigations, and hands-on activities.
Study cell biology, genetics, the history of life, microbiology, botany, zoology, ecology, and human anatomy and physiology, all within a biblical framework.
|Out of class will be mostly reading the chapters and assigned exercises in the workbook, plus a small project each quarter. I will assign 2-3 hours of outside work per week.
|Grades are given weekly. Grades are made up of class participation, daily work, homework, extra credit activities, and chapter quizzes. I use an online grade book that you can view your child's grades or assignments anytime on the Internet. This is a great communication tool to view your child's progress. A "Progress Report" will also be sent home each semester as well as an end of the year cumulative grade.


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Flu epidemic among the United States
Yasmine Castañeda,
January 24, 2018
The flu is spreading throughout the United States and is ranking as one of the most severe influenza seasons in recent years. This year, the flu is now being labeled as an epidemic in terms of scale. Influenza activity has taken off and cases are being reported in all 50 states, Puerto Rico and Gua...
Tinder: Hangout or hookup?
Sarah Williams-Cain,
January 4, 2018
Popular dating app under fire for perpetuating "hookup culture" amongst millennials Millennials are having less sex. Less sex than we have seen in any other generation. However, in an unparalleled coincidence, the number of sexually transmitted disease outbreaks has seen a sharp incline. As a res...


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Google's Project Nightingale: Why Google has access to millions of health care records now
What is it? First reported by the Wall Street Journal, Google is partnering with Ascension. Ascension is the second largest non-profit health system in the U.S. and with the deal struck now allows Google to collect and analyze millions of their users' detailed medical records. The secretive health initiative was monikered 'Project Nightingale' and allows Google access to health records, names, and addresses without telling patients or doctors. Ascension is partnered with over 2,600 hospitals in America.
What Google and Ascension have said: Ascension said in a press release, the deal between the two sides is meant to help Ascension modernize its infrastructure and develop new care models delivered by apps, digital platforms, and other health care services used daily by a lot of users. They are using Google cloud and web services to optimize the digital part of their business, while Google is using Ascension data to help design and build new health-care related software and technology.
Privacy concerns: In the same release, Ascension tried to ease privacy and legal concerns stating, "All work related to Ascension's engagement with Google is HIPAA compliant and underpinned by a robust data security and protection effort and adherence to Ascension's strict requirements for data handling." HIPAA generally allows hospitals to share and sell data to business partners if it is used to help maximize health care service. Google also attempts to ease privacy concerns on their blog stating, "To be clear… patient data cannot and will not be combined with any Google consumer data."
However, the deal has drawn lots of skepticism and criticism from experts who argue this deal is stripping patients of control over their own data. Even Google forthright stating they will not use this data with other data collected by Google has to be met with skepticism because Google is constantly refining and changing their data collecting algorithms. Ascension is not Google's only attempt to gain access to consumers' personal health data though.
Google also recently purchased fitbit for $2.1 billion on November 1. The Fitbit acquisition along with the Ascension deal, make Google's move into healthcare data more questionable and alarming as it seems they have the pieces to map your health records to real-time biometrics. Protecting patients' privacy and information must become more prevalent to health care providers when trying to optimize the efficiency and health care services provided by their businesses.


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Bona Traffic HD is for hardwood floor owners with an uncompromising attitude towards durability, being green and looking beautiful. Significantly outperforms all urethane finishes, including Bona Traffic, in durability at 24 hours.
Bona Traffic HD must be used with Bona Traffic HD hardener. The Traffic/ Hardener must be mixed by shaking it vigorously for one minute. If small quantities are to be mixed, the Traffic HD container must be shaken before decanting product from it. Once mixed, Traffic HD is viable for five hours.
For use ONLY with Bona Traffic HD.
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This article will help you to find the world of video games. Make sure you duck and take cover if reloading your weapon. It's a lot easier to get pwned because they failed to take adequate cover before reloading. You do not want this to occur to you!
More Info at 3ReturnMan.com. Video games are often times very expensive. You can find used video games.
Make sure the screen brighter. If you can't see the game, you will perform much better with a brighter screen. It will make the colors easier to see so your enemies won't see you before they spot you.
Save your games in multiple slots. Sometimes put a new slot. You may want to go a bit in your game and do something differently. You won't have this if you save too often in the same spot.
When buying games for kids, only buy those without violence or adult content.
Take advantage of the parental control settings.Check if the game online. If you can play it online, limit your children's Internet access. You may also need to look at their friend requests and give them a time limit on how much they do not play excessively.
You should keep the games by throwing them out. Many outlets will let you to exchange your old games for a bit of cash or cash. Use this cash from your old games you have traded in to afford brand new ones.
Don't play video games for more than a few hours or less.Gaming is something that some find to be addictive, and there is such a thing as video game addiction, so you have to watch out for that. Limit the amount of time you spend playing to just a couple or three hours each day. If you decide to surpass that limit, then it's best to take plenty of breaks during the day.
Use the best video connection available. A lot of the gaming systems will rely on several cables that can be better than others. What would you to use for the best gaming experience? If those are not an option, S-Video, Composite and RCA connections. Coaxial connections happen to be the most common video games connections, but be aware that this type of connection is not of very high quality. Only use them if you have.
Watch for sales at local establishments to find good deals on video games to give your kids. Some video rental stores are hurting now. If you are lucky, you may find one nearby and get some great video game deals. The games will generally be in good condition, but could use a good cleaning.
Avoid overuse of cheat codes when playing games that depend on your skill as a player. You want to avoid this since it will not enhance your gaming skills at all.
Be adventurous and give lots of different kinds of video games a try! Trying different types of video games can be a fun and easy way to enjoy your gaming experience.
You might be tempted to purchase all the expansions and upgrades from a game as soon as you buy it. This gets expensive very quickly. Before you think about purchasing a new game, do an inventory of what you already have at home.
You can go to sleep when you die! This is a common myth amongst gamers which is even propagated by sites that claim they have the best gaming tips. You need the full eight hours of sleep nightly to perform your mind in great shape so you can strategize correctly.
Video games have been around for some time now. They have undergone a tremendous amount of change. Seeing how video games look now, it's crazy to think what video games will look like in the future. It'll be exciting to find out!


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On last night's show, David "The Menzoid" Menzies joined me to discuss the Ontario Progressive Conservative Party leadership race.
Christine Elliott tweeted out her opposition to a carbon tax, but David explains that the wording of her tweet and her six-figure patronage appointment from Kathleen Wynne indicate she might take a softer stance on the carbon tax once elected.
Caroline Mulroney's campaign has indicated that she will enact Patrick Brown's "People's Guarantee", including the carbon tax. I don't think she would even be a candidate for leader if it wasn't for her last name, and David wasn't impressed after hearing Mulroney interviewed on CFRB.
WATCH my interview to see why David thinks Doug Ford's anti-elite message is his ticket to victory and that opposing the carbon tax is a must for the next PC leader.


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Membership is open to all those interested in working gundogs and prospective members are invited to attend one training session with the club.
Applicants must be proposed and seconded by two current paid up members of the Club and receive the subsequent approval of the Committee.
Annual Subscriptions are £10.00 and are due on April 1st.
PLEASE NOTE if subs are not paid by June 30th a rejoining fee will be required.
Click Here to Download Application Form
New members are subject to a £5.00 joining fee. A re-joining fee of £5.00 will be levied for lapsed membership.
Those accepted for membership after February 1st will be considered as paid members for the year.
Please send application form and cheque for £15.00 to the secretary.
Subsequent subs should be sent direct to the treasurer by cheque.
Alternatively a completed Standing order should be sent direct to your Bank.


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I've been to 12 countries in Europe, including Italy, and this pizza beat every other place i've been to. Service was impeccable as well. Highly recommend.
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Episode 144
September 3rd, 2019
56 mins 21 secs
About this Episode
Ever dreamed of having a Linux laptop in the palm of your hand or in your pocket? That's a reality with SailfishOS. A downloadable Linux operating system for your phone that's as much Linux in spirit as it is in the kernel unlike Android.
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Women for Atlantic Foundation
Supporting and promoting women in Atlantic Canada
There's room for all of us... There's room for everyone. And sometimes its easier to fight for others than it is to fight for yourself...
What We Do
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There is no difference between people... there is often a culture aspect that leads to feeling inadequate, and leading to issues with self esteem... we need to recruit and intervene very aggressively when they get discouraged...
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We all can contribute in different ways. Find your voice, use your skills, and be the change. Make an impact of your own where you can.
Elevate New Brunswick
January 22, 2019