Parasites and animals
The Yangzhou strain of E. necatrix used in this study was originally isolated from a chicken that died from E. necatrix infection in 2009 in Yangzhou, China, as confirmed by microscopic examination and sequence analysis of the internal transcribed spacer region of genomic DNA [24]. This strain has been maintained in our laboratory. The oocysts were periodically propagated in 3–4-week-old chickens. Oocysts were isolated and harvested from the feces by salt flotation and centrifugation, sporulated in vitro at 28°C and stored in 2.5% potassium dichromate solution at 4°C [24].
One-day-old chickens (purchased from the Poultry Institute of the China Agricultural Academy, Yangzhou, Jiangsu, China) were housed in Eimeria-free isolation cages and were provided with complete feed and clean water without anticoccidial drugs. Chicken feces were observed by salt flotation and light microscopy to ensure the absence of Eimeria infection prior to experimental inoculation.
Ten 4-month-old female New Zealand White rabbits (3.5–4 kg) were purchased from the Animal Genetic Engineering Laboratory at Yangzhou University. All rabbits were placed in separate cages and fed under pathogen-free conditions. The animals were allowed access to a standard rabbit diet and water ad libitum in a temperature-controlled room with a 12-h light-dark cycle at 21–23°C and 50–75% relative humidity in the Animal Center of the College of Veterinary Medicine, Yangzhou University.
All animal care and procedures were conducted according to the guidelines for animal use in toxicology. The study protocol was approved by the Animal Care and Use Committee of the College of Veterinary Medicine, Yangzhou University.
Generation Of Polyclonal Antibodies
The recombinant proteins rEnGAM22 and rEnGAM59 were prepared using a previously published method [22, 24]. WFB protein was prepared using the method described below. Mouse anti-rEnGAM59 and WFB pAbs were prepared as described previously [22]. Briefly, the 50 µg proteins, which were resuspended in 50 µL phosphate-buffered saline (PBS) and mixed with 50 µL Quick Antibody-Mouse 3W (Biodragon, Beijing, China), were used to immunize 6-week-old BALB/c mice twice following the manufacturer’s recommendations. Blood was collected 7 days after the second immunization, centrifuged at 1500 ⋅ g for 15 min to isolate the mouse pAbs and stored at -80°C.
Rabbit anti-rEnGAM22 and rEnGAM59 pAbs were generated as follows: the 50 µg proteins, which were resuspended in 100 µL PBS and mixed with 100 µL Quick Antibody-Rabbit 8W (Biodragon), were used to immunize 4-month-old New Zealand rabbits three times following the manufacturer’s recommendations. Blood was then collected 10 days after the third immunization, and the rabbit pAb was separated as previously described and stored at -80°C.
Antibody levels were determined using an enzyme linked immunosorbent assay (ELISA) method as described in a previous study [25]. The results showed that the optical density (OD) values of mouse anti-rEnGAM59 and anti-WFB pAbs were 3.06 (1:200 dilution) and 3.13 (1:200 dilution), whereas the OD values of rabbit anti-rEnGAM22 and anti-rEnGAM59 pAbs were 2.86 (1:200 dilution) and 2.55 (1:200 dilution), respectively. As negative control, the OD values of naïve mice sera were less than 0.13, the OD values of rabbit sera were less than 0.17.(Additional file: Table S1).
Preparation Of Gametocytes
Gametocytes were isolated using previously described methods [26]. Briefly, second-generation merozoites (MZ-2) were obtained from the small intestine of chickens 136 h after oral inoculation with 2.0 × 104 E. necatrix oocysts, and approximately 1.8 × 108 MZ-2 in a volume of 2 mL were injected into the ceca of chickens, as described by McDonald and Rose [27]. At 30 ± 0.5 h after injection with MZ-2, the chickens were sacrificed and the ceca were removed and washed with cold SAC (1 mM phenylmethanesulfonyl fluoride, 1 mg/mL bovine serum albumin [BSA], 170 mM NaCl, 10 mM Tris-HCl pH 7.0, 10 mM glucose and 5 mM CaCl2). Then, the caeca were slit open and the mucosal tissues were scraped and incubated at 37°C in a beaker for 2 h with 0.5 mg/mL of hyaluronidase in SAC. The digested mucosal tissues were filtered through 100, 20 and 17-µm polymon mesh. The filtrate was centrifuged at 3000 ⋅ g for 5 min, and the pellet was resuspended in five volumes of cold erythrocyte lysis buffer (Solarbio, Beijing, China) at 4℃ for 20 min and washed with cold PBS three times by centrifugation. The gametocytes were purified using Percoll (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Finally, the purified gametocytes were counted in a counting chamber, then immediately frozen in liquid nitrogen for future use (Additional file: Fig. S1). The average yield obtained was approximately l06 gametocytes/infected chicken.
Isolation And Purification Of Wfbs
The WFBs of E. necatrix from macrogametocytes were purified as described previously [11] with a minor modification. Briefly, the purified E. necatrix gametocytes (1 × 108 cells) were extracted with 0.1% Saponin in TNEP buffer (10 mM Tris-HCl, pH 7.4; 50 mM NaCl; 2 mM ethylenediaminetetraacetic acid [EDTA]) for 20 min at room temperature and centrifuged at 1000 ⋅ g for 5 min. The resulting pellet was washed three times in TNEP by centrifugation at 3000 ⋅ g for 2 min, and the supernatant was discarded. The pellet was resuspended in five volumes of TNEP and sonicated with an output of 3.0 and duty cycle 30% for 3 s intervals over 4 min in an ice water bath. After filtering the lysates through a 5-µm polymon mesh, the filtrate was added to 5% sodium dodecyl sulphate (SDS; W/V; volume ratio of filtrate to 5% SDS: 4:1), vortexed and centrifuged at 15,000 ⋅ g for 10 min. The WFBs in the pellet were resuspended in 5 mL TNEP buffer, concentrated and purified using a 1000-kDa cut-off Vivaspin 6 centrifugal filter (Sartorius Stedim Biotech, Aubagne, France), centrifuged at 4000 ⋅ g for 20 min and concentrated three times. Then, the concentrated solution was collected, and the purified WFBs were obtained by centrifugation at 15,000 ⋅ g at 4°C for 10 min and stored at 4℃ or frozen immediately in liquid nitrogen for future use.
Identification Of Wfbs
In order to confirm the identity of WFBs, the purified granules were observed by negative-staining electron microscopy, and the proteins were analyzed by SDS-PAGE electrophoresis and Western blot.
Negative-staining electron microscopy
A 5 µl droplet of the purified WFBs was dropped on a carbon-coated copper grid, and after draining the liquid from the side of the copper mesh with filter paper, the sample was stained for 5 min at room temperature with 2% phosphotungstic acid solution (pH 7.0) and dried at room temperature for 2 min after the dye solution was absorbed. The specimens were examined using a Philips Tecnai 12 Biotwin transmission electron microscope (TEM; Philips Electron Optics, Eindhoven, Netherlands), the voltage was adjusted to 80 kV and the WFBs were observed and photographed.
SDS-PAGE and Western blot analysis
The purified gametocytes and WFBs were resuspended in 500 µL Pierce RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA), sonicated with an output of 3.0 and duty cycle 30% for 3 s intervals over 4 min in an ice water bath and centrifuged at 12,000 ⋅ g at 4°C for 10 min. Supernatants were separated and protein concentrations of the lysates were determined using a TaKaRa Bradford Protein Assay Kit (TakaRa, Tokyo, Japan). Total protein lysates (10 µg per lane) were loaded and electrophoresed on a 12% SDS-PAGE gel. The proteins were then visualized by staining with Coomassie Brilliant Blue R (Sigma-Aldrich, St. Louis, MO, USA) or transferred to nitrocellulose membranes (Merck Millipore, Billerica, MA, USA) for 1.5 h at 100 V. After blocking with 3% BSA in tris-buffered saline (TBS) for 12 h at 4°C, the membranes were incubated with mouse anti-rEnGAM59 pAb (1:400 dilution), mouse anti-WFB pAb (1:400 dilution) or rabbit anti-rEnGAM22 pAb (1:400 dilution) at room temperature for 1 h. Prior to washing three times with 0.05% Tween-20/TBS (TBST) over 30 min, the membranes were probed with peroxidase-conjugated Affinipure goat anti-mouse IgG (H + L; 1:10,000; Jackson Immuno Research, PA, USA) o
Localization Of Engam22 And Rengam59 Proteins In Wfbs
In order to confirm the localization of EnGAM22 and rEnGAM59 proteins in WFBs of E. necatrix, indirect immunofluorescence assays (IFAs) were performed on purified WFBs and macrogametocytes as described previously [11, 24, 28]. Briefly, WFBs and macrogametes were placed on 0.1% (v/v) poly-L-lysine-coated glass microscope slides and fixed in methanol (-20°C). After blocking overnight in 5% BSA in PBS (BSA/PBS) at 4°C, the samples were incubated with either rabbit anti-rEnGAM22 pAb (1:100 dilution) and mouse anti-rEnGAM59 pAb (1:100 dilution) or with rabbit anti-rEnGAM22 pAb (1:100 dilution) and mouse anti-WFB pAb (1:100 dilution) for 1 h at 37°C, washed with 0.03% Tween-20/PBS (PBST) three times for 15 min and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100 dilution; MultiSciences Hangzhou, China) and Cy3-conjugated goat anti-mouse IgG (1:100 dilution; Servicebio) in BSA/PBS for 1 h at 37°C, respectively. Before visualization, the samples were rinsed in PBST as described above. Images were obtained using LCM (Leica TCS SP8 STED, Wetzlar, Hessen, Germany). Naïve sera from rabbits and mice were used as a negative control.
Microscopic Examination Of Oocyst Wall Formation Using Lcm
Pathological tissue samples from chickens sacrificed 156 h post-infection were collected as described in our previous study [24]. The purified gametocytes, unsporulated oocysts and tissue sections were used for IFA as described above. However, before visualization, the tissue sections were counter-stained with 4’6-diamidino-2-phenylindole (DAPI, Biyotime, Shanghai, China). Images were obtained using LCM (Leica TCS SP8 STED, Wetzlar). Naïve sera from mice and rabbits were used as a negative control.
Ultrastructural Examination Of Oocyst Wall Formation Using Sem
The purified gametocytes were fixed in 2.5% glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate buffer. The samples were post-fixed in 1% OsO4 (Sigma-Aldrich) for 1 h, dehydrated in a series of graded ethanol solutions (30, 50, 70, 80, 90, 95 and 100%) and dried with a Leica EM CPD300 Automated Critical Point Dryer (Lecia Microsystems GmbH). The samples were glued onto specimen holders, then sputter coated with gold using a Lecia EM SCD500 (Lecia Microsystems GmbH). The samples were examined using a ZEISS Gemini SEM 300 (Carl Zeiss AG, Oberkochen, Germany).