Name: magnetic stirrer method for the detection of trichinella larvae in muscle samples
Uploaded: 2017-03-03T15:00:00.000+00:00
Duration: 9 min 44 s
Description: Watch this Scientific Journal Video about Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples at JoVE.com

We kindly thank Sabine Reckinger for excellent technical assistance and support.

1. Preparations
<ol>
	<li>Sample collection 
	Note: The magnetic stirrer method can be used for single or pooled muscle samples.
	<ol>
		<li>Collect samples from the appropriate predilection sites and in an appropriate amount9. Refer to the country requirements or to the recommendations of ICT, OIE or ISO.
		<ol>
			<li>For the European Union, for example, take a 1 g sample from a diaphragm pillar at the transition to the sinewy part of domestic pigs. Ensure that all muscle samples are free of all fat and fascia.</li>
		</ol>
		</li>
		<li>If the tongue is tested, remove the surface layer prior to testing.</li>
		<li>Take care if samples are frozen, as most Trichinella larvae are not freeze resistant and uncoil after death, are less resistant to digestion and tend to stick to the container walls, resulting in decreased assay sensitivity. 
		Note: If frozen, double the sample size at least and increase the sedimentation time (see below).</li>
	</ol>
	</li>
	<li>Preparation of digestion fluid
	<ol>
		<li>Add 25% HCl (16 &#177; 0.5 mL) to 2 L of tap water preheated at 46 - 48 &#176;C into a 3 L glass beaker. Too low of a temperature results in incomplete digestion; too high of a temperature deactivates the enzymatic properties of pepsin.</li>
		<li>Place a stirring rod in the beaker, and stir the solution on a preheated plate (46 - 48 &#176;C).</li>
		<li>Add 10 &#177; 0.2 g of pepsin (1:10,000 NF, 1:12,500 BP, 2,000 FIP) to the acidic solution. 
		Note: The quality of the pepsin is of utmost importance and enzyme activity must be certified by the producer. For laboratory safety reasons and to maintain the pepsin activity, the sequence of adding digestion fluid components must be adhered to.</li>
	</ol>
	</li>
	<li>Muscle sample preparation
	<ol>
		<li>Chop up to 100 g of muscle samples in a blender or grinder. To facilitate blending, add 2 mL preheated water to the samples and blend at max speed for 2 - 3 s.</li>
		<li>Before the second blending, open the blender and transfer any muscle sample at the top of the blending bowl edge margin to the bottom and repeat blending. Continue blending with bursts of 2 - 3 s until no visible pieces of meat remain. 
		Note: Too little blending may result in incomplete digestion, while too much blending can damage the Trichinella larvae present in the samples6.</li>
	</ol>
	</li>
</ol>
2. Procedure
<ol>
	<li>Muscle sample digestion
	<ol>
		<li>Transfer 1 L of the digestion fluid into a cylinder. Transfer the ground meat into the beaker and disseminate in the digestion fluid with a spoon or fork. Thoroughly rinse the blender lid, blade and blender bowl with 500 mL of the preheated digestion fluid into the 3 L beaker. 
		Note: Incomplete rinsing can result in the loss of sensitivity.</li>
		<li>Cover the beaker with aluminum foil and keep a constant temperature of 44 - 46 &#176;C and monitor with a thermometer.</li>
		<li>Stir the digestion fluid for 30 min to create a deep vortex without splashing until the meat particles disappear. Depending on the type of meat tested (e.g. meat of wild animals), increase the digestion time until a maximum of 60 min.</li>
	</ol>
	</li>
	<li>Filtration and sedimentation of the digestion fluid
	<ol>
		<li>To determine the amount of undigested tissue, adjust the scale to zero, weigh the sieve before use, and note its weight. The sieve should be clean and devoid of any tissue particles, which could hinder the Trichinella larvae reaching the sedimentation funnel. Check that the separation funnel is leveled vertically.</li>
		<li>After digestion, carefully pour the digestion fluid through a 180 &#181;m sieve into a separation glass funnel. The width of the separation glass funnel should not be larger than 55% of the length to allow good larva sedimentation. Take care to avoid any overflow.</li>
		<li>To avoid loss of sensitivity, thoroughly rinse the glass beaker and the sieve with approximately 200 mL of tap water from a squeeze wash bottle and transfer the rinsing water into the sedimentation glass funnel. Take care to carefully rinse the edges of the 3 L beaker. Lift the sieve and rinse the area under the sieve thoroughly.</li>
	</ol>
	</li>
	<li>Determination of undigested tissue amount 
	Note: Due to errors in the execution of the method, muscle tissue can remain on the sieve, as well fascia and connective tissue which are indigestible. The amount of undigested tissue is determined during the first 10 min sedimentation step (see 2.5). This allows approximately 30 min for the sieve to dry, as residual water can influence the determined weight of the undigested tissue.
	<ol>
		<li>Place a paper towel on a scale, and weigh the sieve with the undigested tissues.</li>
		<li>Subtract the sieve weight before filtration from the sieve weight with undigested tissues. The digestion process is considered satisfactory if not more than 5% of the starting sample weight remains on the sieve (i.e. 5 g/100 g starting material).</li>
		<li>If the amount of undigested tissue exceeds 5%, repeat the test using fresh muscle samples. If the remaining tissue is predominantly comprised of undigested muscle tissue and fresh samples are not available, digest the undigested remains again and examine in addition to the original samples.</li>
	</ol>
	</li>
	<li>Sedimentation of the digestion fluid
	<ol>
		<li>Keep the filtered digestion fluid in the separatory funnel for 30 min. If left undisturbed, the Trichinella larvae will settle at the bottom of the funnel.</li>
		<li>If frozen samples are used, increase the sedimentation time to a maximum of 60 min.</li>
		<li>Once sedimentation is complete, fully open the stopcock of the separatory funnel to let at least 40 mL of digestion fluid quickly run off into a measuring cylinder (80 mL if muscle samples had been previously frozen).</li>
		<li>First, let half of the fluid into the cylinder and then fully open the stopcock in the opposite direction to allow 10 mL of digestion fluid to pass. Finally, open the stopcock in the opposite direction for another 10 mL. This procedure ensures that Trichinella larvae are not trapped in the stopcock. 
		Note: Sufficient speed is needed to avoid larvae remaining in the separatory funnel, decreasing sensitivity.</li>
	</ol>
	</li>
	<li>Sedimentation of the digestion fluid in the cylinder
	<ol>
		<li>Leave the sediment in the cylinder for 10 min to allow larvae to sediment. Remove 30 mL supernatant by suctioning with a pipette without disturbing the 10 mL sediment.</li>
		<li>To reduce the amount of tissue fibers in the sample, add 30 mL tap water to the sediment and leave for another 10 min. Repeat until the liquid is clear.</li>
		<li>Remove supernatant and transfer the 10 mL washed sediment to a gridded Petri dish or larval counting basin. Rinse the cylinder with 10 mL tap water and then add the water to the sample. 
		Note: As large amounts of debris in the sample can prevent the identification of the Trichinella larvae (Figure 1), further washing steps should be performed, if required.</li>
	</ol>
	</li>
	<li>Microscopic examination
	<ol>
		<li>Use a trichinoscope or a stereo-microscope with a sub-stage transmitted light source of adjustable intensity at a 15 to 20X magnification to examine the samples immediately after the washing steps.</li>
		<li>After pouring the sample into the gridded Petri dish, examine the dish/basin systematically grid by grid. If no suspect structures are found, gently move the fluid in the gridded Petri dish or larval counting basin in a circular fashion to allow any Trichinella larvae, which were potentially overlooked, to accumulate in the center of the Petri dish. Repeat the examination.</li>
		<li>If a suspect structure is found, increase magnification to 40 - 100X. Remove Trichinella larvae from the dish/basin with a pipette and collect in a tube with 90% ethanol.</li>
		<li>After the complete dish has been examined, repeat the procedure, starting from the gentle shaking of the dish/basin. Repeat the procedure at least three times or until no more larvae are found.</li>
		<li>Count the number of larvae. Correlate to the amount of muscle tissue tested and present as larvae per gram (lpg).</li>
		<li>If too many larvae are present in the digestion fluid to determine the larval number reliably, return the fluid containing the larvae to the measuring cylinder, rinse with tap water from a squeeze wash bottle, and leave larvae to settle to the bottom.</li>
		<li>After a 10 min sedimentation time, reduce the supernatant to a defined volume (e.g. 10 mL). To determine the exact volume, transfer the supernatant into a new cylinder with a pipette.</li>
		<li>Suspend larvae homogenously in the fluid by vigorous shaking. During the shaking motion, add 12 drops of 20 &#181;L larval suspension onto a Petri dish.</li>
		<li>Examine each drop by microscope. Determine the number of larvae in 240 &#181;L and delete the highest and lowest count. Extrapolate the number of larvae to the total volume (e.g. 10 mL) of supernatant. 
		Note: The quality of the stereo-microscope is of paramount importance for the identification of Trichinella larvae.</li>
		<li>When larvae are collected in a tube by a pipette, check carefully that the larvae did not stick on the pipette wall but are transferred to the tube. 
		Note: Positive Trichinella findings from pooled samples must be traced back from the pool to the carcass of origin. Here, the pool size should progressively be decreased until the positive carcass is identified. The sample size can also be increased during this process to increase sensitivity. Where examination of a collective sample produces a positive or uncertain result, a further 20 g sample is taken from each pig. The 20 g samples from five pigs are pooled and examined using the described method. In this way samples from 20 groups of five pigs will be examined. When Trichinella is detected in a pooled sample from five pigs, 20 g samples are collected from the individual pigs in the group and each is examined separately until the positive carcass of origin is detected.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gottstein, B., Pozio, E., N&#246;ckler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology,+Diagnosis,+Treatment,+and+Control+of+Trichinellosis.">Epidemiology, Diagnosis, Treatment, and Control of Trichinellosis.</a> Clin. Microbiol. Rev. 22, 127-145 (2009).</li><li>Pozio, E., Murrell, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematics+and+epidemiology+of+Trichinella.">Systematics and epidemiology of Trichinella.</a> Adv. Parasitol. 63, 367-439 (2006).</li><li>9 CFR Part 318.10 - Prescribed treatment of pork and products containing pork to destroy trichinae. U.S. Government Publishing Office Available from: <a target="_blank" href="https://www.gpo.gov/fdsys/pkg/CFR-2016-title9-vol2/pdf/CFR-2016-title9-vol2-sec318-10.pdf">https://www.gpo.gov/fdsys/pkg/CFR-2016-title9-vol2/pdf/CFR-2016-title9-vol2-sec318-10.pdf</a> (2012)</li><li>Gamble, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Commission+on+Trichinellosis:+recommendations+on+methods+for+the+control+of+Trichinella+in+domestic+and+wild+animals+intended+for+human+consumption.">International Commission on Trichinellosis: recommendations on methods for the control of Trichinella in domestic and wild animals intended for human consumption.</a> Vet. Parasitol. 93 (3-4), 393-408 (2000).</li><li>Gajadhar, A. A., Forbes, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internationally+recognized+quality+assurance+system+for+diagnostic+parasitology+in+animal+health+and+food+safety,+with+example+data+on+trichinellosis.">An internationally recognized quality assurance system for diagnostic parasitology in animal health and food safety, with example data on trichinellosis.</a> Vet. Parasitol. 103, 133-140 (2002).</li><li>N&#246;ckler, K., Kapel, C. M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Detection+and+surveillance+for+Trichinella:+meat+inspection+and+hygiene,+and+legislation.">Chapter 3, Detection and surveillance for Trichinella: meat inspection and hygiene, and legislation.</a> FAO/WHO/OIE Guidelines for the surveillance, management, prevention and control of trichinellosis. , 69-98 (2007).</li><li>Boch, J., Schnieder, T., Supperer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Veterin&#228;rmedizinische Parasitologie. , (2006).</li><li>Despommier, D. D., M&#252;ller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stichosome+and+its+secretion+granules+in+the+mature+muscle+larva+of+Trichinella+spiralis.">The stichosome and its secretion granules in the mature muscle larva of Trichinella spiralis.</a> J. Parasitol. 62 (5), 775-785 (1976).</li><li>Marucci, G., Interisano, M., La Rosa, G., Pozio, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+nematode+larvae+different+from+those+of+the+Trichinella+genus+detected+by+muscle+digestion.">Molecular identification of nematode larvae different from those of the Trichinella genus detected by muscle digestion.</a> Vet Parasitol. 194, 117-120 (2013).</li><li>Forbes, L. B., Gajadhar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+Trichinella+digestion+assay+and+an+associated+sampling+and+quality+assurance+system+for+use+in+testing+pork+and+horse+meat.">A validated Trichinella digestion assay and an associated sampling and quality assurance system for use in testing pork and horse meat.</a> J. Food Prot. 62, 1308-1313 (1999).</li><li>Malakauskas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+epidemiology+of+Trichinella+spp.+in+three+Baltic+countries:+Lithuania,+Latvia,+and+Estonia.">Molecular epidemiology of Trichinella spp. in three Baltic countries: Lithuania, Latvia, and Estonia.</a> Parasitol. Res. 100 (4), 687-693 (2007).</li><li>Kapel, C. M., Webster, P., Gamble, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+distribution+of+sylvatic+and+domestic+Trichinella+larvae+in+production+animals+and+wildlife.">Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife.</a> Vet. Parasitol. 132 (1-2), 101-105 (2005).</li><li>Interisano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+latex+agglutination+test+for+the+detection+of+Trichinella+infections+in+pigs.">Validation of a latex agglutination test for the detection of Trichinella infections in pigs.</a> Vet. 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The authors declare that they have no competing financial interests.

Although the magnetic stirrer method can be easily performed, not strictly adhering to technical details leads to insufficient sensitivity, thus endangering consumer health. The critical control points have been highlighted in the text above. In addition, the use of appropriate equipment is crucial for the successful outcome of the test. All vessels should be made of glass and pipette tips coated with silicon to reduce adherence of larvae to the surface.
The sensitivity of the digestion method depends on the larval density in the muscles and the amount of muscle sample tested. For a larval density of 3 - 5 lpg of muscle tissues, a sensitivity of 100% was reported, but below 1 lpg the sensitivity dropped to 40%13. Depending on the tested host animal species, the sensitivity of the test can be improved by increasing the amount of sample used. The infection burden in wildlife is usually low, therefore larger samples sizes are used14. Predilection sites also vary among host animals. Here, the lower digestibility of some muscle tissues such as the tongue needs to be taken into account, which can also result in larger sample sizes15.
Further, it is important to understand that the magnetic stirrer method does not include internal controls. Therefore, quality assurance management from sampling to documentation is essential to obtain accurate and precise results. The quality of laboratory performance to detect Trichinella larvae in muscle samples should be monitored by regular participation of each analyst in proficiency tests.
Further limitations of the method are that the final identification stage of muscle larvae by microscopy is highly subjective and heavily dependent on the knowledge of larval morphology by the examining analyst.
An interesting alternative method to circumvent this problem is the magnetic stirrer method/on filter isolation followed by larvae detection by a latex agglutination test. However, according to EU legislation this method is only considered equivalent for the testing of meat of domestic swine but not for animals which are at higher risk of infection such as wild boar4. The identification of the larval antigen with monoclonal antibodies makes the test more objective, but denies the laboratory the possibility of determining the number of larvae per gram of meat16.
Further variations of the magnetic stirrer method include the mechanically assisted pooled sample digestion method/sedimentation technique, the mechanically assisted pooled sample digestion method/on filter isolation technique, and the automatic digestion method for pooled samples of up to 35 g technique. These techniques are all based on the artificial digestion of the meat and the visualization and counting of Trichinella larvae, but differ in the equipment used for the filtration and sedimentation steps.
The isolated larvae can be further identified at the species level by a molecular test (e.g., multiplex PCR)17. According to EU legislation, all positive samples must be forwarded to the national reference laboratory or to EURLP for the Trichinella species determination.

Nematodes of the genus Trichinella can be detected in striated muscles of carnivore and omnivore mammals, birds, and reptiles worldwide. These zoonotic nematodes can reach human beings when raw or semi-raw meat and meat derived products from swine, horse, and game animals are ingested. This zoonosis can be a serious disease in humans characterized by pathognomonic signs and symptoms, e.g. diarrhea, fever, periorbital edema and myalgia and possible complications such as myocarditis, thromboembolic disease and encephalitis1.
Trichinella spiralis is the most widespread etiological agent of Trichinella infection in wild and domestic animals and causes most of the human infections worldwide. In Europe, North and West Africa, and Western Asia, Trichinella britovi is another source of human infections. In addition, ten other taxa are less commonly reported as the causative agents of the human disease and are found in different regions of the world, usually in wild animals2. In addition to wild animals such as wild boar, bears, walruses and badgers, pork represents the most important source of human infection worldwide.
The best way to prevent trichinellosis is to refrain from eating raw meat products and to cook any meat, especially game meat to safe temperatures (&#62; 65 &#176;C for 1 min, i.e. to change the meat color from pink to brown in the core of the meat product)3. The European Union and USDA have specified freezing and cooking times and temperatures for pork products that can be used to kill T. spiralis larvae in meat4,5. Furthermore, recommendations for the inactivation of Trichinella in meat and meat products were published by the International Commission on Trichinellosis6. In Europe, in addition to the introduction of controlled housing conditions in commercial swine herds where the risk of Trichinella infection is negligible, the prevention of human trichinellosis is based on the laboratory examination of muscle samples from susceptible animals4.
For food safety purposes, digestion assays are the only reliable procedures for the direct detection of Trichinella larvae in meat3,7. Even if there are several variations of the digestion assay, the magnetic stirrer method is the internationally accepted reference method3,4,7. This assay is based on the detection of Trichinella larvae in striated muscle tissues. After the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps, Trichinella larvae are identified by microscopy. Depending on trade obligations and national legislation, there are a multitude of small variations in the general protocol of the magnetic stirrer method. Here, technical details are based on EU requirements4, ISO specifications8, ICT guidelines6, and the experience of the European Union Reference Laboratory for Parasites (EURLP) and the German Reference Laboratory for Trichinella.

<table><tbody><tr><td>Knife, Scissors, Tweezers</td><td></td><td></td><td>Cutting samples and removing non-digestible tissue</td></tr><tr><td>Blender&amp;nbsp;</td><td></td><td></td><td>With a sharp chopping blade</td></tr><tr><td>Magnetic stirrer</td><td></td><td></td><td>With thermostatically controlled heating plate</td></tr><tr><td>Stirring Rod</td><td></td><td></td><td>Teflon-coated, approximately 5 cm long</td></tr><tr><td>Conical glass separation funnel</td><td></td><td></td><td>Capacity of at least 2 L, preferably fitted with Teflon safety plugs. The width should not be larger than 55% of the length to allow a good larva sedimentation.</td></tr><tr><td>Stands, rings and clamps</td><td></td><td></td><td></td></tr><tr><td>Sieve</td><td></td><td></td><td>Mesh size 180 microns, external diameter 11 cm, with stainless steel mesh</td></tr><tr><td>Funnel</td><td></td><td></td><td>Internal diameter not less than 12&amp;nbsp;cm, to support the sieves</td></tr><tr><td>Glass beaker</td><td></td><td></td><td>Capacity 3 L</td></tr><tr><td>Glass measuring cylinder</td><td></td><td></td><td>Capacity 50 to 100 mL, or centrifuge tube</td></tr><tr><td>Stereo-microscope</td><td></td><td></td><td>With a substage transmitted light source of adjustable intensity or trichinoscope with a horizontal table</td></tr><tr><td>Petri dishes</td><td></td><td></td><td>9 cm diameter, marked on their undersides into 10 &amp;times; 10 mm square examination areas using a pointed instrument or a larval counting basin for the tricinoscope</td></tr><tr><td>Aluminium foil</td><td></td><td></td><td>Alternatively a lid to cover the beakers</td></tr><tr><td>25% hydrochloric acid</td><td></td><td></td><td></td></tr><tr><td>Pepsin</td><td></td><td></td><td>Strength: 1:10,000 NF (US National Formulary) corresponding to 1:12,500 BP (British Pharmacopoeia) and to 2,000 FIP (F&amp;eacute;d&amp;eacute;ration internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/mL</td></tr><tr><td>Ethanol</td><td></td><td></td><td>70-90% ethyl alcohol</td></tr><tr><td>Tap water</td><td></td><td></td><td>Heated to 46-48 &amp;deg;C before use</td></tr><tr><td>Balance</td><td></td><td></td><td>Accurate to at least 0.1 g</td></tr><tr><td>Metal tray</td><td></td><td></td><td>Capacity 10 to 15 L, to collect the remaining digestive juice</td></tr><tr><td>Pipettes and pipette holder</td><td></td><td></td><td>Different sizes (1, 10 and 25 mL)</td></tr><tr><td>Thermometer</td><td></td><td></td><td>Accurate to 0.5 &amp;deg;C, temperature range at least from 20 &amp;deg; C to 70&amp;nbsp;&amp;deg;C</td></tr><tr><td>Siphon</td><td></td><td></td><td>For tap water</td></tr></tbody></table>

magnetic stirrer method for the detection of trichinella larvae in muscle samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

After digestion, the shape of the infective muscle larvae can vary, making identification more difficult. Typical forms include tightly coiled larvae, lightly coiled larvae or c-shaped or completely uncoiled larvae (Figures 2B - 2C). The number of larvae found per gram can vary considerably and can range from one larva to a few hundred larvae.
The morphology of the infective muscle larva is shown in Figure 2A. Trichinella larvae are 0.7 to 1.5 mm long and approximately 0.3 mm in width. The esophagus is narrow and has a slightly rounded end. The cuticle is smooth. In the anterior half of the body cavity, the stichosome, a structure constituted of a long slender tube surrounded by a row of 45 to 55 large cells (stichocytes), can be observed. The rectum is also rounded without any projections or appendages10,11.
Other structures, besides Trichinella larvae, can be found after muscle digestion. Some examples are shown in Figures 3A - 3F. Wild animals are often infected with other parasites or the muscle samples available for testing are contaminated during the evisceration process by animate or inanimate structures/organisms. The length of the larvae, the appearance of the anterior and posterior ends and the occurrence of the stichosome help to discriminate between the different nematode genera12.
<img alt="Trichinella larva microscope images, labeled A and B, showcasing detailed morphology for parasitology study." src="/files/ftp_upload/55354/55354fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Microscopic Examination of Trichinella Larvae After (A) Insufficient and (B) Sufficient Rinsing Steps. Scale bars indicate 100 µm. <a href="http://cloudflare.jove.com/files/ftp_upload/55354/55354fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy images showing nematode anatomy; labeled structures include stichosome, rectum, esophagus." src="/files/ftp_upload/55354/55354fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Morphology of Infective Trichinella Larvae Showing the Rectum, Stichosome and Esophagus of the Larva (A). Possible shapes of the muscle larvae after digestion showing (B) tightly coiled and lightly coiled moving larvae and (C) uncoiled larvae. Scale bars indicate 100 µm. <a href="http://cloudflare.jove.com/files/ftp_upload/55354/55354fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopic images of various helminths; labeled A-F; parasitology study; identification analysis." src="/files/ftp_upload/55354/55354fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Examples of Incidental Findings after Digestion of Muscle Samples with the Magnetic Stirrer Method. (A) bristle-like hair of earth worm found in wild boar; (B) Alaria alata from wild boar; (C) muscle fiber from wild boar; (D) Metastrongylus sp. from wild boar; (E) plant fiber found in wild boar (F): Toxocara sp. larva from wild boar. Scale bars indicate 100 µm. <a href="http://cloudflare.jove.com/files/ftp_upload/55354/55354fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples at JoVE.com

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

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Research

JoVE Journal

Immunology and Infection

28.7K Views.  Federal Institute for Risk Assessment. The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Video: Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Biology

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Developmental Biology

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the development of several animal models of these neuromuscular disorders has provided the scientific community with different therapeutic scenarios aimed at delaying or reversing the progression of these conditions. By taking advantage of the retrograde machinery of neurons, one of these approaches has been to target skeletal muscles in order to shuttle therapeutic genes into corresponding spinal cord motor neurons. Although once promising, the success of such gene delivery approach has been hampered by the sub-optimal number of transduced motor neurons it has so far shown to yield. Motor end plates (MEPs) are highly specialized regions on the skeletal musculature that are in direct synaptic contact to the spinal cord &#945; motor neurons. In this regard, it is important to note that, so far, the efforts to retrogradely transfer genes into motor neurons were made without reference to the location of the MEP region in the targeted muscles. Here, we describe a simple protocol 1) to reveal the exact location of the MEPs on the surface of skeletal muscles and 2) to use this information to guide the intramuscular delivery and subsequent optimal retrograde transport of retrograde tracers into motor neurons. We hope to utilize the results from these tracing experiments in further studies into investigating retrograde transport of therapeutic genes to spinal cord motor neurons through the targeting of MEPs.

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Diseases affecting the integrity of spinal cord motor neurons are amongst the most debilitating neurological conditions. Over the last decades, the ...

Neuroscience

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

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Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells