Name: diagnosis of ecto- and endoparasites in laboratory rats and mice
Uploaded: 2011-09-06T12:00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice at JoVE.com

1. Endoparasite examination (Table 1)
1. Perianal tape test (also see section 5; these are usually performed at the same time)
<ol>
	<li>Remove a length of clear, not frosted, cellophane tape from a dispenser. Tape should be long enough to handle by one end without touching the middle (approximately 5 cm). It may be easier to dispense several lengths at one time, attaching them to the edge of a clean work surface and using as needed. </li>
	<li>Lift a mouse from its cage and place on the cage lid, holding it by the tail. Perform this action in a laminar flow cabinet or biosafety cabinet if the health status of the animal requires it.</li>
	<li>Restrain the mouse by the tail, lifting its hind legs from the cage. Grasp the end of the tape between thumb and forefinger, then apply the middle of the tape firmly to the mouse's perineum, including the perianal area several times. Hair should be seen to be adherent to the tape for the assay to be considered successful.</li>
	<li>Place the mouse back in the cage. </li>
	<li>Place a drop of mineral oil on a labeled clean glass slide, apply the tape to the slide, then another drop of mineral oil. Cover with a glass cover slip.</li>
	<li>Read the microscope slide using the 10x and 40x objectives on a light microscope. The perianal tape test is best at detecting Syphacia eggs, although other parasite eggs are sometimes found.</li>
</ol>
2. Fecal flotation
<ol>
	<li>Assemble flotation solution, a flat-bottomed vial (pill vial or fecal flotation device such as Ovatector), petri dish, cover slip, microscope slide, and applicator/stirring sticks. Floatation solution, such as Fecasol, should have a specific gravity of 1.20-1.30 and may be made from various sodium salts, sugar, zinc sulfate, or purchased commercially. (Table 2)</li>
	<li>Collect 2-5 fecal pellets from the cage or fresh from the animal(s) into the flotation chamber. If feces are extremely dry, either due to age or species producing the fecal matter, moistening the feces with 500 &mu;l of 0.9% saline may be beneficial.</li>
	<li>Place the vial in the petri dish to protect the working surface from overflow of dissolved feces. Add a small volume of floatation medium and mash and stir thoroughly. No large pieces of material should remain. Continue to add flotation medium until a meniscus forms above the edge of the vial.</li>
	<li>Place the cover slip on the meniscus and incubate at room temperature for 15 minutes. Parasite eggs and some protozoan oocysts will rise to the top and adhere to the cover slip.</li>
	<li>After incubation, lift and invert the cover slip. Place the cover slip on a glass microscope slide.</li>
	<li>Examine the slide using the 10x and 40x objectives under a light microscope.</li>
	<li>Although low-tech and easy to perform, in general, this technique is not recommended for mice or rats. As a rule, it is more suitable for animals that produce a larger volume of feces.</li>
</ol>
3. Fecal concentration and centrifugation
<ol>
	<li>Assemble flotation solution, centrifuge tube, cover slip, microscope slide, and applicator/stirring sticks and tube caps. Flotation solution should have a specific gravity of 1.18-1.30 and may be made from various sodium salts, sugar, zinc sulfate, or purchased commercially. (Table 2)</li>
	<li>Collect 2-10 fecal pellets from the cage or fresh from the animal(s) into a collection tube. If feces are extremely dry, either due to age or species producing the fecal matter, moistening the feces with 500 &mu;l of 0.9% saline or with the flotation solution you are using may be beneficial.</li>
	<li>Mix sample in an appropriate flotation solution within a glass centrifuge tube. Mechanical agitation with a vortexer may be used to mix samples. If a vortexer is used, snap caps should be placed on the tops of the tube to prevent spillage and cross contamination. Routinely, samples are prepared in 1.18 specific gravity zinc sulfate, however other solutions may be used in addition (in a separate preparation tube) or in substitution. </li>
	<li>Add additional flotation solution to each tube to form a slight positive meniscus on each tube. Apply a plastic cover slip to each tube, and ensure that full contact with tube lip is made. Place tube(s) into the centrifuge.</li>
	<li>Centrifuge at approximately 616-760 RCF for 10 minutes. If cover slips are lost or broken during the centrifugation process, a new cover slip can be placed on the sample tube and the tube can be gently tipped so that the meniscus touches the new cover slip. No additional centrifugation is necessary.</li>
	<li>Remove cover slip from centrifuge tube and place on a labeled, clean glass microscope slide. If multiple centrifugation solutions were used to evaluate one fecal sample, two cover slips may be placed on the same slide.</li>
	<li>Stain the slide with iodine. This allows for easier identification of cysts. </li>
	<li>Examine the slide using the 10x and 40x objectives on a light microscope.</li>
</ol>
4. Direct examination of intestines for helminths and protozoa
<ol>
	<li>Place the euthanized mouse or rat carcass in dorsal recumbency on a clean dissection board or similar work surface.</li>
	<li>Using forceps, lift the abdominal wall at the genital area. Using scissors, carefully incise the ventral abdominal wall from the genital area to the base of the ribcage removing both skin and muscle and exposing the intestines. Remove the intestines, beginning at the duodenum (the segment of the intestine beginning at the exit of the stomach) and continuing to the descending colon (the segment of the intestine which ends at the anus and usually contains formed feces).</li>
	<li>Place intestines in a 100 ml Petri dish. Collect a portion of cecum and duodenum from the euthanized animal and place on a dissecting board. Incise each intestinal segment lengthwise to expose the mucosa.</li>
	<li>Using scissors, cut the remaining intestines into small sections. Add enough tap water to the dish to barely submerge the collected tissue.</li>
	<li>Incubate the sample mixture at 35-40&deg;C in a laboratory oven or incubator for a minimum of 10 minutes. This will liberate and expose luminal helminths. While the sample is incubating, carry out the following steps.</li>
	<li>Place two drops of 0.9% saline side by side on a single labeled slide, to allow for preparation of samples from two intestinal segments (duodenum and cecum).</li>
	<li>Heat sterilize and cool an inoculating loop or equivalent. </li>
	<li>Scrape the mucosa of the duodenum and place the scrapings on the left hand side of the slide (side closest to the frosted edge).</li>
	<li>Scrape the mucosa of the cecum and place the scrapings from on the right hand side of the slide.</li>
	<li>Top the scrapings with a cover slip. Examine prepared slide using the 40x objective under a phase contrast microscope); increase magnification as needed for identification. If parasites are detected, identify based on morphology. </li>
	<li>The dish contents will be ready for examination by this point. Examine the dish contents under a dissecting microscope. Use a probe or applicator stick as necessary to move contents within the dish to complete a thorough examination. If pinworms are present they will appear as small, white, hair-like worms. If tapeworms are present, they will appear as segmented, flat worms (larger than the thread-like pinworms).</li>
	<li>If helminths are detected or suspected, collect the specimen using a small pair of forceps.</li>
	<li>Mount the specimen on a labeled, clean glass slide in a drop of paraffin or mineral oil and place a cover slip on top of the specimen. Examine the slide using the 10x and 40x objectives on a light microscope.</li>
</ol>
2. Ectoparasite examination (Table 3)
5. Fur pluck examination for ectoparasites (tape test)
<ol>
	<li>Remove a length of clear, not frosted, cellophane tape from a dispenser. Tape should be long enough to handle by one end without touching the middle (approximately 5 cm). It may be easier to dispense several lengths at one time, attaching them to the edge of a clean work surface and using as needed. </li>
	<li>Lift a mouse or rat from its cage and place on the cage lid, holding it by the tail. Perform this action in a laminar flow cabinet or biosafety cabinet if the health status of the animal requires it.</li>
	<li>Restrain the mouse or rat. Grasp the fur with hemostats and gently pluck fur from the mouse's scapular area, ventral cervical region, axillary area, inguinal area, and dorsal rump. Place the fur on the tape. Hair should be seen to be adherent to the tape for the assay to be considered successful. Place the mouse or rat back in its cage.</li>
	<li>Place a drop of mineral oil on a labeled clean glass slide; apply the tape, then another drop of mineral oil. Cover with a glass cover slip. </li>
	<li>Read the microscope slide using the 10x and 40x objectives under a light microscope.</li>
	<li>This examination is best for detecting fur mites such as Radfordia, Myobia, and Myocoptes.</li>
</ol>
6. Skin scrape
<ol>
	<li>Assemble the following materials: animal to be tested, mineral oil, microscope slide, cover slip, scalpel, and scissors. If this test is to be performed on live animals, they should be anesthetized before beginning.</li>
	<li>Sample the dorsum near the base of the tail and the temporal region of the head. Alternatively, skin lesions and/or other sites may be scraped. </li>
	<li>Deeply scrape the skin with the scalpel blade in the opposite direction of hair growth, to erode the epidermis. Trimming the hair coat prior to scraping may improve screening sensitivity by reducing visual obstruction (excess hair) on the slide.</li>
	<li>Place a drop of oil on the slide. Apply the sample to the oil drop by wiping the blade (with sample attached) on the surface of the slide. Add additional oil to the slide if necessary, and top with a cover slip.</li>
	<li>Read the microscope slide using the 10x and 40x objectives under a light microscope.</li>
	<li>The skin scrape is generally used to detect Demodex (and dermatophytic fungi).</li>
</ol>
7. Direct examination of pelage
<ol>
	<li>Place the euthanized mouse or rat on the stage of a dissecting microscope.</li>
	<li>Examine the hairs of the pelt at approximately 10X using an applicator stick or similar instrument to part the hair and observe the base of the hair shaft.</li>
	<li>Examine the cranial region, between the eyes and the pinnae, between the pinnae, between the scapulae, under the jaw, and the inguinal and axillary areas. Alternatively, the whole carcass may be examined. </li>
	<li>Collect any ectoparasites or suspicious material seen using a small pair of forceps. Ectoparasites may often look like dandruff or a yellow waxy buildup at the base of a hair shaft or directly on the skin.</li>
	<li>Mount the specimen on a clean glass slide in a drop of paraffin or mineral oil and place a cover slip on top of the specimen. Examine the slide using the 10x and 40x objectives under a light microscope.</li>
</ol>
3. Representative Results: 
See attached files identifying the following parasites: (Note: these procedures will detect any egg, helminth, or cyst present in the feces or on the skin and fur; only a few of these are listed below)
Endoparasites:
<table border="0">
	<tbody>
		<tr>
			<td>Syphacia muris (egg, worm)</td>
			<td>Chilomastix bettencourti</td>
		</tr>
		<tr>
			<td>Syphacia obvelata (egg, worm)</td>
			<td>Hexamastix muris</td>
		</tr>
		<tr>
			<td>Aspiculuris tetraptera (egg, worm)</td>
			<td>Retortamonas sp.</td>
		</tr>
		<tr>
			<td>Rodentolepis nana (egg, worm)</td>
			<td>Giardia spp.</td>
		</tr>
		<tr>
			<td>Tritrichomonas muris</td>
			<td>Spironucleus muris</td>
		</tr>
		<tr>
			<td>Entamoeba muris</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>		
Ectoparasites:
<table border="0">
	<tbody>
		<tr>
			<td>Myocoptes musculinis</td>
			<td>Radfordia affinis</td>
		</tr>
		<tr>
			<td>Myocoptes musculinis</td>
			<td>Radforida ensifera</td>
		</tr>
		<tr>
			<td>Myobia musculi</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Tape test</td>
			<td>Fecal flotation</td>
			<td>FCC</td>
			<td>Direct exam1</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Protozoa</td>
			<td>--</td>
			<td>+</td>
			<td>++</td>
			<td>+++</td>
			<td>+++/NA2</td>
		</tr>
		<tr>
			<td>Metazoa</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pinworm</td>
			<td>+/-3</td>
			<td>+/-4</td>
			<td>+4</td>
			<td>+++</td>
			<td>+++</td>
		</tr>
		<tr>
			<td>Tapeworm5</td>
			<td>--</td>
			<td>+</td>
			<td>++</td>
			<td>+++</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Other roundworms5</td>
			<td>--</td>
			<td>+</td>
			<td>+++</td>
			<td>+++</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>
1. This method requires euthanasia of the animal. 
2. There are not PCR detection methods currently available for every protozoan. 
3. This method is most appropriate to detect Syphacia spp. 
4. This method will be more likely to detect Aspiculuris, and less likely to detect Syphacia. 
5. Tapeworms and roundworms other than pinworms are very rare in modern laboratory mice and rats.
Table 1. Class of endoparasite and appropriate detection method. Some methods will require euthanasia of the animal. NA indicates method is not currently available for these parasites.+ indicates suitability of the method for detection of the parasite in question, and &#x2013; indicates that the method is not recommended for that parasite.
<table border="1">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Specific gravity</td>
			<td>Ingredients per 1L H2O</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>1.20</td>
			<td>311 g sodium chloride</td>
		</tr>
		<tr>
			<td>Sodium nitrate</td>
			<td>1.20</td>
			<td>338 g sodium nitrate</td>
		</tr>
		<tr>
			<td>Sodium nitrate</td>
			<td>1.30</td>
			<td>616 g sodium nitrate</td>
		</tr>
		<tr>
			<td>Sugar</td>
			<td>1.20</td>
			<td>1170 g sucrose1</td>
		</tr>
		<tr>
			<td>Sheather's sugar</td>
			<td>1.27-1.30</td>
			<td>1563 g sucrose1</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>1.18</td>
			<td>493 g zinc sulfate</td>
		</tr>
	</tbody>
</table>
1. These solutions require refrigeration or the addition of 9 ml phenol as a preservative.
Table 2. Fecal flotation solutions (from Smith et al.)
<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>Fur pluck (tape test)</td>
			<td>Skin scrape1</td>
			<td>Direct exam1</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Lice</td>
			<td>--</td>
			<td>--</td>
			<td>++</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Mites</td>
			<td>+</td>
			<td>+</td>
			<td>+++</td>
			<td>+++/NA3</td>
		</tr>
		<tr>
			<td>Fleas4</td>
			<td>--</td>
			<td>--</td>
			<td>+</td>
			<td>NA</td>
		</tr>
		<tr>
			<td>Ticks4</td>
			<td>--</td>
			<td>--</td>
			<td>++</td>
			<td>NA</td>
		</tr>
	</tbody>
</table>
1. This method requires anesthesia if it is to be performed on a live animal. 
2. This method requires euthanasia of the animal. 
3. There are not PCR detection methods currently available for every species of mite. 
4. Fleas and ticks are extremely rare in modern laboratory animal facilities.
Table 3. Class of ectoparasite and appropriate detection method. Some methods will require euthanasia of the animal, and other methods will require anesthesia to perform them in live animals. NA indicates method is not currently available for these parasites. NA indicates method is not currently available for these parasites.+ indicates suitability of the method for detection of the parasite in question, and &#x2013; indicates that the method is not recommended for that parasite.

<ol>
	<li>Baker, D. G., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> The Mouse in Biomedical Research: Diseases. Diseases Vol. 2, (2007).</li><li>Baker, D. G., Baker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11.">Chapter 11.</a> Flynn's Parasites of Laboratory Animals. , 303-398 (2007).</li><li>Owen, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasites of Laboratory Animals. 12, (1992).</li><li>Pritchett, K. R., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+22.">Chapter 22.</a> The Mouse in Biomedical Research: Diseases. Diseases Vol. 2, (2007).</li><li>Smith, P. H., Wiles, S. E., Malone, J. B., Monahan, C. M., Baker, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1.">Chapter 1.</a> Flynn's Parasites of Laboratory Animals. , 1-13 (2007).</li><li>Wasson, K., Fox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+21.">Chapter 21.</a> The Mouse in Biomedical Research: Diseases. Vol. 2, 517-550 (2007).</li></ol>

The authors are all employees of Charles River, a supplier of diagnostic tests and reagents, including the tests described above.

When working in a laboratory, safety should always be a concern. Remember to wear appropriate protective equipment when working with animals and to clean your workstation with disinfectant before and after. These methods are primarily designed to find any parasites of laboratory rodents in the locations examined, i.e., they can detect exotic or exceedingly rare parasites as well as the more common pinworms and fur mites. Although they are equally applicable to other species, wild rodents may have additional parasites in locations such as liver, subcutis and brain, not evaluated by the above methods.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cutting board</td>
			<td>Thermo Electron Corp</td>
			<td>Cat #36114</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>ROBOZ</td>
			<td>RS-5910, G204</td>
			<td>23mm blades, 3.5&#x201D; length, straight</td>
		</tr>
		<tr>
			<td>Medium scissors</td>
			<td>ROBOZ</td>
			<td>RS-6808, G207</td>
			<td>5&#x201D;</td>
		</tr>
		<tr>
			<td>Forceps-Curved</td>
			<td>ROBOZ</td>
			<td>RS-8254 (M1/21004)</td>
			<td>4.5&#x201D;, serrated, slight curve</td>
		</tr>
		<tr>
			<td>Forceps-Microdissecting </td>
			<td>ROBOZ</td>
			<td>RS-5238</td>
			<td>Hudson-(EWALD)</td>
		</tr>
		<tr>
			<td>Forceps-Tissue Forceps</td>
			<td>ROBOZ</td>
			<td>RS-8160</td>
			<td>Rat tooth</td>
		</tr>
		<tr>
			<td>Metal probe</td>
			<td>VWR</td>
			<td>Cat#25778-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Vantage</td>
			<td>V97-48</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Applicator sticks</td>
			<td>Puritan</td>
			<td>6in Applicators, Ref#807</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus</td>
			<td>SZ51, Schott, ACE1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>VWR</td>
			<td>100mm, Cat#3401PDNL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>VWR</td>
			<td>Micro cover glass, Cat#48366-067</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>VWR</td>
			<td>VistaVision microscope slides Cat#16004-368</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inoculating loop</td>
			<td>VWR</td>
			<td>Cat#50815-040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>Model#BX41TF</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory oven</td>
			<td>Quincy Lab Inc.</td>
			<td>Model 10 Lab Oven</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-12R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>VWR</td>
			<td>Mini-Vortexer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Test tube</td>
			<td>Kimble-Chase</td>
			<td>15 ml disposable centrifuge tube, Cat#73790-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>VWR</td>
			<td>Plastic microscope cover slips, 22mm, Cat#48376-049</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>White caps</td>
			<td>VWR</td>
			<td>Cat#60869-089</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Rowley biochemical institute</td>
			<td>Cat#SO-364</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>Sigma Aldrich</td>
			<td>Cat#1000917519, Z4750-500G </td>
			<td>&nbsp;</td>
		</tr>

		<tr>
			<td>Sucrose</td>
			<td>Mallinckrodt Chemicals</td>
			<td>Cat#8360-06</td>
			<td>&nbsp;</td>
		</tr>

		<tr>
			<td>Phenol</td>
			<td>EMD</td>
			<td>Cat#PX0510-1</td>
			<td>&nbsp;</td>
		</tr>

		<tr>
			<td>Cellophane tape</td>
			<td>Staples</td>
			<td>Invisible tape, Cat#504712</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Mallickrodt </td>
			<td>Cat#6358</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

diagnosis of ecto- and endoparasites in laboratory rats and mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

Watch this Scientific Journal Video about Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice at JoVE.com

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

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Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...