Theileria parva-infected ticks
The R. appendiculatus colony at the Animal Disease Research Unit of the United States Department of Agriculture’s Agricultural Research Service (USDA-ARS) was started from 50 male and 50 female ticks received in 2013 from Dr. Ivan Morrison at the Roslin Institute in Edinburgh, Scotland. At that time the colony had been maintained at the Roslin Institute for 25 years; ticks for the colony at Roslin were originally collected from Muguga [15]. A calf was subcutaneously inoculated with a stabilate of R. appendiculatus salivary glands infected with T. parva Muguga sporozoites, as described previously [16, 17]. The animal was monitored for clinical signs of disease and by polymerase chain reaction (PCR) to confirm infection with T. parva. Genomic DNA was extracted to determine infection using Tp104-PCR. Forward (5′-CGC CTG AGC CAA AAG CTA GTA-3′) and reverse (5′-TTC GAT GGC CTC GGT GAT T-3′) primers were designed to amplify a fragment of 149 base pairs. Reactions were performed in 25 μl containing 2 μl of template DNA, 0.4 µM of each primer, and 12.5 µl of RedTaq (Sigma-Aldrich, St. Louis, MO, USA) under the following conditions: one cycle at 95 °C for 3 min, 35 cycles of 95 °C for 30 s, 56 °C for 20 s and 72 °C for 30 s, with a final extension at 72 °C for 5 min. Amplicons were analyzed by 2% agarose gel electrophoresis. Amplicons were sequenced to verify parasite specificity (Eurofins Genomics, Louisville, KY, USA).
When the animal became febrile, developed mild peripheral lymphadenopathy, and was PCR-positive for T. parva in peripheral blood, approximately 1000 R. appendiculatus nymphs were applied under a cloth patch on the back of a calf and allowed to feed to repletion. Replete nymphs were collected, washed in tap water, and incubated at 26 °C and 93% relative humidity (RH) to molt to adults. Molted adult ticks were maintained at 15 °C and 93% RH and used in the in vitro tick feeding system within 2 to 4 months.
This study was approved by the Institutional Animal Care and Use Committee of the University of Idaho (Moscow, ID, USA), in accordance with the recommendations of the US Animal Welfare Act (United States Code, Title 7, Chapter 54, sections 2131–2159) and Animal Welfare Regulations (Code of Federal Regulations, Title 9, Chapter 1, Subchapter A, parts 1–4). The T. parva-infected animal developed severe ECF and was euthanized by intravenous injection of sodium pentobarbital (Fatal-Plus, Vortech Pharmaceuticals, USA).
Detecting ticks infected with T. parva
Infected adult ticks were fed in the in vitro tick feeding system to allow T. parva sporozoite development in salivary gland acini cells. Adult ticks were fed blood at a constant 37 °C temperature. After 5 days of feeding, 23 ticks were dissected, salivary glands harvested, and genomic DNA extracted. The salivary gland infection rate and number of parasites were determined by PCR, as described above, and Tp104 quantitative PCR (Tp104-qPCR). Briefly, a primer set, forward (5′CAG ATG GAA GTG AAG TGT 3′) and reverse (5′ TAA ATG AAC AAG TGA TGC 3′), was designed to amplify a 101-base-pair fragment. A standard curve was constructed by amplification of 106, 105, 104, 103, and 102 plasmid copies of the Tp104 gene as previously described [18]. The amplification reaction was performed in three technical replicates in a final volume of 20 µl using 0.4 µM of each primer, 1.5 µl of a template, and 10 µl of SsoFast™ EvaGreen® Supermix. The qPCR conditions consisted of an initial cycle at 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The number of T. parva sporozoites is presented as the average log of the triplicate values. CFX Manager™ software (Bio-Rad) was used to acquire the qPCR data. Standard deviations were calculated using Microsoft Excel.
Pathogen localization within salivary glands
The presence of T. parva in acini cells was examined using immunohistochemistry (IHC) as previously described [19]. After sporozoite stimulation in the in vitro tick feeding system, infected ticks were fixed with 10% formalin and embedded in paraffin, and sequential 4 µm sections were deparaffinized in Clear-Rite and hydrated using an ethanol (100–70%) gradient. Sections were treated with citrate solution, pH 6 (Zymed, Carlsbad, CA, USA) for antigen retrieval and steamed for 30 min. The sections were stained using 2 µg/ml of anti-PIM (IgG2a) or anti-P67 (IgG1) monoclonal antibodies [20]. Monoclonal antibodies anti-ANA8 (IgG1) [21] and anti-F16 (IgG2a) [22] were used as negative isotype controls. To detect primary antibody binding, sections were incubated with horseradish peroxidase-labeled anti-mouse immunoglobulin (Dako Corp., Carpinteria, CA, USA) and 3-amino-9-ethylcarbazole containing hydrogen peroxide. Sections were counterstained with Mayer’s hematoxylin. Sections were examined using a Nikon Eclipse microscope.
Isolation of in vitro secreted T. parva sporozoites
Fifty R. appendiculatus adults (25 males and 25 females) infected with T. parva were applied to the silicone membrane of the in vitro tick feeding system as described previously [23]. The ticks were allowed to feed initially on uninfected bovine blood at a packed cell volume of 10% containing 1× antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA). Adult ticks were fed blood at a constant 37 °C temperature. After 21 h, the blood receptacle was washed three times with distilled water, once with 70% ethyl alcohol, and twice with phosphate-buffered saline (PBS) to remove blood residue. Four milliliters of cell-free complete RPMI medium at 37 °C containing 10% fetal bovine serum, 24 mM of HEPES, 2 mM of l-glutamine, and 10 µg/ml of gentamicin was added to the blood receptacle. Ticks were fed for 3 h. Subsequently, the medium was collected, and sporozoites recovered by centrifugation at 5000×g for 2 min. Blood was replaced to the blood receptacle, and ticks were fed for 21 h. The process was repeated for six consecutive days. Genomic DNA was extracted to determine the number of secreted sporozoites by Tp104-qPCR as described above.
Infectivity of secreted T. parva sporozoites
Infectivity of secreted T. parva sporozoites was determined by incubating isolated parasites with bovine PBMC from an uninfected animal in vitro [24, 25]. Sixty ml of whole bovine blood from an uninfected animal was collected into EDTA and centrifuged at 1200×g for 10 min at room temperature. The buffy coat was recovered and suspended in Hank's Balanced Salt Solution. The cells were overlaid onto Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 900×g for 30 min. Cells were collected from the Histopaque interface, washed three times, suspended in 5 ml of complete RPMI 1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% calf bovine serum, 20 mM HEPES buffer (Gibco), 50 µM β-mercaptoethanol (Gibco), 2 mM L-glutamine (Gibco), and 50 µg/ml gentamicin (Gibco). PBMC were infected as previously described [24] by exposing 106 bovine cells per well with ~ 105 secreted T. parva sporozoites and incubating at 37 °C with 5% CO2. Secreted T. parva sporozoites were collected on day 5 post-tick attachment.
Theileria parva-exposed lymphocytes were analyzed at different time points after in vitro infection using flow cytometry and immunocytochemistry (ICC). For flow cytometry, harvested cells were washed with PBS, fixed, and permeabilized with fixation/permeabilization solution (BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit) (BD BioSciences) for 20 min at 4 °C. The fixed cells were washed twice with BD Perm/Wash buffer (BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit) (BD BioSciences). Cells were stained on ice with 2.5 μg/ml anti-T. parva PIM monoclonal antibody [20] conjugated with DyLight 650 for 30 min in the dark. Autologous uninfected lymphocytes exposed to 5 µg/ml Concanavalin A (ConA) (Sigma-Aldrich) and stained with anti-PIM monoclonal antibody were used as a negative control. Stained cells were washed twice with Perm/Wash buffer and acquired with a Guava easyCyte flow cytometer using InCyte guavaSoft 3.1.1 (Luminex). The data were analyzed with FCS Express version 6 (De Novo Software, Pasadena, CA, USA) to provide the percentage of total cells by quadrant.
For ICC, exposed cells were collected and cytospun at 1000 rpm for 3 min onto positively charged glass slides (Fisher Scientific, Waltham, MA, USA). Fixed cells were probed with 1 μg/ml of anti-PIM or isotype control anti-F16 monoclonal antibody [22]. Horseradish peroxidase-labeled anti-mouse immunoglobulin and 3-amino-9-ethylcarbazole containing hydrogen peroxide was used to detect immunoglobulin binding. Lymphocytes were counterstained with Mayer's hematoxylin (Sigma-Aldrich). Slides were examined using a Nikon Eclipse microscope.
Titration of secreted T.
parva sporozoite infectivity
Tenfold serial dilutions of secreted T. parva sporozoites were performed, and sporozoites combined immediately with 106 bovine PBMC as described above [24, 25]. PBMC were incubated at 37 °C with 5% CO2 and T. parva-infected lymphocytes analyzed by flow cytometry at different time points after in vitro infection and ICC as described above.