Research reported in this publication was supported by Elite Researcher Grant Committee under award number 958680 from the National Institute for Medical Research Development (NIMAD), Tehran, Iran.

This study was approved by the National Institute for Medical Research Development and the Research Ethics Review Committee, No. 958680. Lentiviruses are classified as BSL-2 organisms; hence, all laboratory culture procedures in this protocol were carried out using sterile laboratory practices and conducted under a laminar flow hood according to NIH guidelines. Figure 1 demonstrates a schematic presentation of the study protocol for the different E. granulosus stages.
1. Collecting hydatid cysts
<ol>
	<li>Collect liver hydatid cysts from naturally infected sheep routinely slaughtered at an abattoir.</li>
	<li>Completely sterilize the cyst surface using sterile gauze and 70% alcohol before aspirating the protoscoleces (PSCs).</li>
	<li>Aspirate 20-50 mL of hydatid fluid out into sterile 50 mL conical tubes.</li>
	<li>After draining, blot out the cyst with a sharp blade, transfer the germinal layer into a sterile 50 mL conical tube, and shake it for a short period (~2 min) to increase the collection of PSCs. Transfer the germinal layer to a new 1.5 mL tube for molecular characterization.</li>
	<li>Wash the PSCs 3-5x with phosphate-buffered saline (PBS).</li>
	<li>Observe the PSCs in 0.1% eosin for 5 min to determine the PSC viability under a light microscope. Use PSCs with at least 95% viability for culture.</li>
	<li>Treat the PSC precipitate with 2 mL of pepsin (2 mg/mL, pH 2) for 15-20 min.</li>
	<li>To isolate individual PSCs, resuspend the PSC sediment in PBS and pass it through two layers of sterile gauze into a new sterile 50 mL conical tube. 
	NOTE: Calculate the average of three counts on 50 &#181;L of PSC sediments using a light microscope. For subsequent trials, use the estimated value to determine PSCs/&#181;L. Each hydatid cyst isolate should be characterized based on nucleotide polymorphisms by molecular methods, such as PCR-sequencing17 or high-resolution melting curve analysis18.</li>
</ol>
2. Biphasic cultivation of PSCs of E. granulosus to obtain adult worms
NOTE: Isolated PSCs should be cultured in sterile conditions under a laminar flow cabinet Class II. Prepare the solid and liquid phases of the biphasic culture medium separately before proceeding to the next steps19.
<ol>
	<li>Prepare the biphasic culture medium to consist of two phases.
	<ol>
		<li>For the solid phase, add 10 mL of bovine serum to a 25 mL flask and leave it to coagulate at 76 &#176;C for 20-30 min.</li>
		<li>For the liquid phase (modified CMRL), mix 100 mL of heat-inactivated fetal calf serum (FCS), 36 mL of 5% yeast extract, 5.6 mL of 30% glucose (in distilled water), 1.4 mL of 5% dog bile in PBS, 20 mM HEPES buffer, and 10 mM NaHCO3 in 260 mL of CMRL-1066 medium. Finally, add penicillin (100 IU/mL) and streptomycin (100 mg/mL) to the mixture.</li>
	</ol>
	</li>
	<li>Add 10 mL of the liquid phase medium to each 25 cm2 culture flask.</li>
	<li>Add ~5,000 PSCs to the culture flask and transfer the flask to the CO2 incubator (37 &#176;C, 5% CO2).</li>
	<li>After 24-48 h, transfer the PSCs to a new flask with the solid phase (bovine serum) and add 1 mL of the same fresh liquid phase medium per flask. Transfer the flasks into the CO2 incubator (37 &#176;C, 5% CO2).</li>
	<li>Every 5-7 days, replace the medium with fresh liquid phase medium based on changes in the color of the culture medium and the estimated proportion of differentiated PSCs. 
	NOTE: Protoscoleces respond quickly to environmental changes after being cultured, and most of them undergo differentiation within 2-3 weeks. Due to the consumption of nutrients and protoscoleces&#39; growth and development, the pH of the culture medium changes, and its color shifts toward orange and yellow.</li>
</ol>
3. Monophasic cultivation of PSCs of E.&#160;granulosus
<ol>
	<li>Ensure that monophasic cultivation is done 24-48 h before transduction.
	<ol>
		<li>Culture ~5,000 PSCs in a 25 cm2 culture flask with RPMI and 10% fetal bovine serum (FBS).</li>
		<li>Transfer the flasks into the CO2 incubator (37 &#176;C, 5% CO2) until use.</li>
	</ol>
	</li>
</ol>
4. Cell culture for virus production and preparation
NOTE: Human embryonic kidney 293T (HEK293T) cells were obtained for vector production from the Pathology and Stem Cell Research Center, Kerman University of Medical Sciences.
<ol>
	<li>Transfer 5 &#215; 105 HEK293T cells to a 25 cm2 flask containing 5 mL of DMEM with 10% FBS, 100 U/mL penicillin, and 100 &#181;g/mL streptomycin.</li>
	<li>Incubate the HEK293T cells at 37 &#176;C in 5% CO2 and passage them in the complete culture medium every 48 h. Finally, use low-passage HEK293T cells for transduction20.</li>
</ol>
5. Production and preparation of the virus with the third-generation lentiviral vectors
NOTE: The lentivirus vector pCDH513b (transfer vector) and PLPII, PLPI, and PMD2G (helper vectors) were used to express the GFP reporter gene. See the Table of Materials and Supplemental Figure S1.
<ol>
	<li>Day 1:
	<ol>
		<li>After trypsinizing the HEK293T cells21 in 6-well culture plates, seed 7 &#215; 104 cells per well with fresh antibiotic-free DMEM containing 10% FBS.</li>
		<li>Use cells at 50%-60% confluency for transduction by the calcium phosphate method.</li>
	</ol>
	</li>
	<li>Day 2:
	<ol>
		<li>Replace the cell culture medium 2-3 h before the experiment with fresh antibiotic-free DMEM containing 2% FBS.</li>
		<li>In a 1.5 mL tube, mix the third-generation lentiviral plasmids, including 7 &#181;g/&#181;L pCDH513b (transfer vector), 4 &#181;g/&#181;L PLPII, 4 &#181;g/&#181;L PLPI, and 2 &#181;g/&#181;L PMD2G (helper vectors).</li>
		<li>Add HEPES buffer (0.28 M NaCl, 0.05 M HEPES, and 1.5 mM Na2HPO4; optimal pH range, 7.00-7.28) to the mixture to a volume of 422 &#181;L.</li>
		<li>Add 16 &#181;L of 1% Tris-EDTA (TE) buffer to the mixture.</li>
		<li>Add 62 &#181;L of 2.5 mM calcium chloride to the tubes and mix well.</li>
		<li>Add 500 &#181;L of 2x HEPES-buffered saline (HBS) to the mixture, drop by drop, within 1-2 min using a Pasteur pipette. Mix gently until a semi-opaque solution is obtained, and incubate the mixture for 20 min at room temperature.</li>
		<li>Add the mixture to each plate slowly and distribute it with slow circular motions.</li>
		<li>Incubate the plates at 37 &#176;C in a 5% CO2 incubator and replace the medium after 4-6 h with antibiotic-free DMEM containing 10% FBS.</li>
	</ol>
	</li>
	<li>Day 3:
	<ol>
		<li>Confirm successful transient transduction and cell viability using an inverted fluorescence microscope.</li>
	</ol>
	</li>
	<li>Day 4:
	<ol>
		<li>Harvest viruses from the culture medium by collecting each well&#39;s supernatant and store them at -70 &#176;C until use. 
		NOTE: Harvested lentiviruses could be concentrated and titrated for precise transfection22.</li>
	</ol>
	</li>
</ol>
6. Transient transduction of different stages of E.&#160;granulosus with the virus
<ol>
	<li>Day 1:
	<ol>
		<li>Use a 12-well culture plate for gene transfer. Culture 5 &#215; 103-5 &#215; 104 HEK293T cells in triplicate, with complete DMEM medium as the internal reference.</li>
		<li>Culture 150 fresh PSCs in triplicate in RPMI and 10% FBS.</li>
		<li>Culture 30 strobilated worms in triplicate in DMEM containing 10% heat-inactivated FBS. 
		NOTE: All transduction media must be antibiotic-free. Keep three non-treated control wells for HEK293T cells, PSCs, and strobilated worms in the process.</li>
		<li>Prepare a mixture of 1 &#215; 106 viruses with 4 &#181;g/mL transfection reagent (see the Table of Materials) to transduce the HEK293T cells and different stages of the worm.</li>
		<li>Add the mixture to each plate slowly and distribute it with slow circular motions. Incubate the plates for 4-6 h in a CO2 incubator (37 &#176;C, 5% CO2). Replace the medium for each sample with the same complete culture medium to minimize the toxic effects of the transfection reagent.</li>
	</ol>
	</li>
	<li>Day 2:
	<ol>
		<li>Repeat steps 6.1.4-6.1.5 to increase the efficiency of transient transduction for the HEK293T cells, PSCs, and strobilated worms.</li>
		<li>Incubate all the plates for 24-48 h in a CO2 incubator with the same culture conditions based on the type of cell media.</li>
	</ol>
	</li>
	<li>Day 3:
	<ol>
		<li>Check whether the transduction is successful by using fluorescence microscopy. 
		NOTE: Consider using further confirmatory assays like PCR/qPCR20,23 or Western blotting24 techniques in the gene transfer procedure.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

Understanding the molecular basis of nematodes and Platyhelminthes biology is crucial to understanding the pathogenicity of zoonotic parasites27. The lack of an effective gene evaluation system is a major obstacle to the direct interpretation of functional genetics of cestode parasites, including Echinococcus species12,27. The present study demonstrates the excellent potential of lentivirus in E. granulosus transient transduction.
Lentiviral transduction combines the simplicity of use and speed of transient transduction with the strong expression of stable cell lines to deliver transgenes to mammalian cells28. The major goal of this work was to illustrate the process of lentiviral vector transduction, which will be critical in future Echinococcosis research. 
Specific critical points must be noted in this protocol. Strict sterile workflow conditions should be observed due to the omission of antibiotics in the culture medium, without which bacterial and fungal contamination can be expected after 24 h. Microbial contamination reduces the motility and viability of the parasites, and both transfected and control samples will be lost after 72 h.
Selecting the appropriate stages in the life cycle of Platyhelminthes and suitable culture conditions are key to successful gene transduction12,27. We tested our lentiviral transduction method in the metacestode and strobilated stages. The results showed that strobilated worms were more responsive to transient transduction of the gene than the PSCs, which may be the result of the extensive tegumental structure in the strobilated forms. However, due to the nature of lentiviral transduction, the fluorescence intensity in the multicellular PSCs and strobilated worms is typically much lower than in monolayer HEK293T cells26.
The potential of using Echinococcus as a new model in flatworms for studying biological phenomena has long been discussed. Therefore, a better understanding of Echinococcus biology as a model organism can be useful for expanding stem cell research and regulating the gene expression and evolutionary biology of other platyhelminths8,29,30,31,32. There is an urgent need for applied genetic research at the genomic and transcriptomic levels for Echinococcus and various parasitic and free-living flatworm model systems. Therefore, developing an effective gene transfer process for tapeworms paves the way for new techniques, such as virus-based gene knock-in or CRISPR-Cas-mediated gene editing32,33. This work presents lentiviral transduction in a tapeworm model and demonstrates promising outcomes, with potential implications for flatworm biology. However, more in-depth studies are required to improve the method and develop the applicability of this technique for future experiments.

Cystic echinococcosis (CE) is one of the most important helminth diseases caused by Echinococcus granulosus, a small tapeworm within the family Taeniidae1,2. Extensive studies on immunodiagnostic and vaccine development for E. granulosus have been carried out. However, inadequate knowledge about the molecular basis of parasite biology poses major limitations in the diagnosis, management, and prevention of hydatid disease3,4,5,6.
In recent years, due to the development of genome sequencing and transcriptomic methods, a wide range of molecular studies have been conducted on flatworms by several research groups7,8,9. However, in the world of parasites, advances in gene transfer technology in parasitic flatworms are still limited compared with the highly reproducible transient transduction methods developed for some protozoa10,11,12.
The use of viral delivery systems has emerged as an essential tool for transgene delivery and gene/protein investigations over the last two decades13. Lentivirus infects both dividing and non-dividing cells, thus making it possible to infect postmitotic cells14,15,16. Recent evidence indicates that using a lentivirus-based transduction system in mammalian cells offers the potential to overcome most of the limitations of previous knock-in/knock-down techniques. The design and construction of expression lentiviral vectors with appropriate molecular markers, such as GFP expression, have been described previously16. Therefore, we evaluate lentiviral transient transduction of a GFP reporter gene in the protoscoleces and strobilated worms of E. granulosus.

<table><tbody><tr><td>12-well culture plates</td><td>SPL Life Sciences</td><td>30012</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; culture flask</td><td>SPL Life Sciences</td><td>70325</td><td></td></tr><tr><td>6-well culture plates</td><td>SPL Life Sciences</td><td>30006</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C4901-500G</td><td>Working concentration: 2.5&amp;nbsp;mM</td></tr><tr><td>CMRL 1066 medium</td><td>Thermo Fisher Scientific</td><td>11530037</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>memmert</td><td>ICO150</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270-1KG</td><td></td></tr><tr><td>DMEM</td><td>Life Technology</td><td>12100046</td><td></td></tr><tr><td>Dog bile</td><td></td><td></td><td>Isolated from a euthanized dog and sterilized by 0.2 &amp;mu;m syringe filter</td></tr><tr><td>Eosin Y</td><td>Sigma-Aldrich</td><td>E4009-5G</td><td>prepare 0.1% of Eosin for working exclusion test</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>DNAbiotech</td><td>DB9723-100ml</td><td>Heat inactivation&amp;nbsp;of&amp;nbsp;FBS (30 min in 40 &amp;deg;C)</td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>DNAbiotech</td><td>DB9724-100ml</td><td>Heat inactivation&amp;nbsp;of&amp;nbsp;FCS (30 min in 40 &amp;deg;C)</td></tr><tr><td>HEK293T cells</td><td>BONbiotech</td><td>BN_0012.1.14</td><td>Human embryonic kidney 293T</td></tr><tr><td>HEPES buffered saline (HBS)</td><td>Sigma-Aldrich</td><td>51558-50ML</td><td>2x concentrate</td></tr><tr><td>Inverted fluorescence microscope</td><td>OLYMPUS</td><td>IX51</td><td></td></tr><tr><td>Penicillin</td><td>Sigma-Aldrich</td><td>P3032-10MU</td><td>Working concentration: 100 IU/mL</td></tr><tr><td>Pepsin</td><td>Roche</td><td>10108057001</td><td>Working concentration: 2 mg/mL, pH 2</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>DNAbiotech</td><td>DB0011</td><td>This reagent solve in less than 1 min in D.W</td></tr><tr><td>Polybrene (Transfection reagent)</td><td>Sigma-Aldrich</td><td>TR-1003-G</td><td></td></tr><tr><td>RPMI medium</td><td>BioIdea</td><td>BI-1006-05</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761-1KG</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma-Aldrich</td><td>S9137-25G</td><td>Working concentration: 100 &amp;mu;g/mL</td></tr><tr><td>Third-generation lentiviral plasmid (pCDH513b)</td><td>SBI System Biosciences (BioCat GmbH)</td><td>CD513B-1-SBI</td><td>Transfer vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td></tr><tr><td>Third-generation lentiviral plasmid (pLPI and pLPII)</td><td>Invitrogen (Life Technologies)</td><td>K4975-00</td><td>Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td></tr><tr><td>Third-generation lentiviral plasmid (pMD2G)</td><td>Addgene</td><td>Plasmid 12259</td><td>Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran)</td></tr><tr><td>Tris/EDTA Buffer (TE)</td><td>DNAbiotech</td><td>DB9713-100ml</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T9935-50MG</td><td>1x working solutions (pH 7.4&amp;ndash;7.6)</td></tr></tbody></table>

transient transduction of the strobilated forms of echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Here, we describe a rapid and efficient transient transduction technique in E. granulosus by using third-generation lentiviral vectors. We cultured PSCs in a biphasic culture medium to obtain strobilated worms, as described previously25,26. Protoscoleces develop into strobilated worms after 6 weeks in vitro. Different stages of E. granulosus were observed in the biphasic culture medium, including invaginated PSCs (Figure 2A), evaginated PSCs (Figure 2B), and strobilated worms with first and third proglottid formation (Figure 2C-E). Protoscoleces and the strobilated worms obtained from monophasic and biphasic cultures, respectively, were transfected with GFP-expressing lentiviruses (Figure 3). To avoid autofluorescence effects, microscopic observations were compared with the background fluorescence in all three groups of samples, and all samples above this background level were considered transfected.
We adjusted the field illumination and lowered the shutter to prevent any possible autofluorescence emission in the control samples for each treatment. We then checked lentiviral vector-treated samples, wherein green light emission against a black field was considered effective transient transduction. HEK293T cells-the transient transduction process control-clearly expressed GFP (Figure 3D). The adult worms expressed GFP most distinctly in the tegumental layer (Figure 3F); however, PSCs demonstrated a somewhat lower level of GFP expression after 48 h. Some cyst fluid residues and/or germinal layer debris around the PSCs caused some fluorescence in the background in the transfected samples. The intensity of fluorescence in HEK293T cells and strobilated worms increased after 24 h and 48 h (minor changes were observed in PSCs).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62783/62783fig01.jpg" /> 
Figure 1: A schematic presentation of the study protocol. Abbreviation: PSCs = protoscoleces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62783/62783fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62783/62783fig02.jpg" /> 
Figure 2: The different stages of Echinococcus granulosus in biphasic culture medium. (A) Invaginated PSCs, (B) evaginated PSCs, (C, D) worms in the process of strobilation, (E) strobilated worms with third proglottid formation, (F) worms in different stages of strobilation in the culture medium. Scale bars = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62783/62783fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62783/62783fig03.jpg" /> 
Figure 3: Transient transductionof different stages of Echinococcus granulosus and the control cell line in light and fluorescence microscopy. (A,D) HEK293T cells as transduction process control, (B, E) protoscoleces, and (C, F) strobilated worms. (G, H, I) Mixed light and florescence microscopy. Scale bars = 50 &#181;m, 100 &#181;m, and 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62783/62783fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Map of pCDH-CMV-MCS-EF1-GreenPuro cDNA cloning and expression vector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62783/Supplementary Figure S1 (Map of pCDH).pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Transient Transduction of the Strobilated Forms of Echinococcus granulosus at JoVE.com

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

transient-transduction-strobilated-forms-echinococcus

Nanyang Technological University

Research

JoVE Journal

Genetics

2.9K Views.  Kerman University of Medical Sciences. Voorbijgaande transactie van de gestroopte vormen van Echinococcus granulosus. We beschrijven een snelle transiënte transductietechniek in verschillende ontwikkelingsstadia van Echinococcus granulosus met behulp van lentivirale vectoren van de derde generatie. Cystische echinokokkose of hydatidziekte is een van de belangrijkste zoönotische parasitaire ziekten veroorzaakt door Echinococcus granulosus, een kleine lintworm in de darm van honden.Er is dringend behoefte aan toegepast gene...

Video: Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Immunology and Infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Biology

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes. A comprehensive understanding of Rickettsia characteristics would ultimately improve clinical diagnosis, assessment, and treatment with improved control and prevention of the disease. 
Functional studies of rickettsial genes are crucial for understanding their role in pathogenesis. This paper describes a procedure for the electroporation of the Rickettsia parkeri strain Tate's Hell with the shuttle vector pRAM18dSFA and the selection of transformed R. parkeri in tick cell culture with antibiotics (spectinomycin and streptomycin). A method is also described for the localization of transformed R. parkeri in tick cells using confocal immunofluorescence microscopy, a useful technique for checking transformation in vector cell lines. Similar approaches are also suitable for the transformation of other rickettsiae.

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

Ultrastructural Expansion Microscopy in In Vitro Life Cycle of Trypanosoma cruzi

Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment.
Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques. 
We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

This study shows a detailed protocol to perform ultrastructure expansion microscopy in three in vitro life cycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. We include the optimized technique for cytoskeletal proteins and pan-proteome labeling.

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

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