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.
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.
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 (> 65 °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.
1. Preparations
2. Procedimento
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.
Figure 1: Microscopic Examination of Trichinella Larvae After (A) Insufficient and (B) Sufficient Rinsing Steps. Scale bars indicate 100 µm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
We kindly thank Sabine Reckinger for excellent technical assistance and support.
Knife,Scissors, Tweezers | Cutting samples and removing non-digestible tissue | ||
Blender | With a sharp chopping blade | ||
Magnetic stirrer | With thermostatically controlled heating plate | ||
Stirring Rod | Teflon-coated, approximately 5 cm long | ||
Conical glass separation funnel | Capacity of at least 2 litres, preferably fitted with Teflon safety plugs. The width should not be larger than 55% of the length to allow a good larva sedimentation | ||
Stands, rings and clamps | |||
Sieve | Mesh size 180 microns, external diameter 11 cm, with stainless steel mesh | ||
Funnel | Internal diameter not less than 12 cm, to support the sieves | ||
Glass beaker | Capacity 3 litres | ||
Glass measuring cylinder | Capacity 50 to 100 ml, or centrifuge tube | ||
Stereo-microscope | With a substage transmitted light source of adjustable intensity or trichinoscope with a horizontal table | ||
Petri dishes | 9 cm diameter, marked on their undersides into 10 × 10 mm square examination areas using a pointed instrument or a larval counting basin for the tricinoscope | ||
Aluminium foil | Alternatively a lid to cover the beakers | ||
25 % hydrochloric acid | |||
Pepsin | Strength: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml | ||
Ethanol | 70-90% ethyl alcohol | ||
Tap water | Heated to 46-48 °C before use | ||
Balance | Accurate to at least 0.1 g | ||
Metal tray | Capacity 10 to 15 litres, to collect the remaining digestive juice | ||
Pipettes and pipette holder | Different sizes (1, 10 and 25 ml) | ||
Thermometer | Accurate to 0.5 °C, temperature range at least from 20 ° C to 70 ° C | ||
Siphon | For tap water |