Trichinella spp.: Etiological agents of human trichinosis of food origin. Detection in animals and food: Trichinoscopy; Artificial digestion-concentration ; Identification of species (PCR and sequencing)
Nematodes of the genus Trichinella spp. are the causative agents of trichinosis, a zoonotic parasitosis of food origin caused by the consumption of raw or undercooked meat infected with viable larvae located in muscle cells.
This parasitosis constitutes a risk for human health, and also causes considerable economic losses in the world food production. Although trichinosis is endemic in many regions of the world, the predominant impact of human diseases is mainly related to acute outbreaks after the consumption of infected raw meat products. The European Food Safety Authority (EFSA) classifies the presence of Trichinella as a medium risk to public health in relation to the consumption of pork meat. In addition, trichinosis appears in the list of diseases of the Terrestrial Animal Health Code of the World Organization for Animal Health (OIE), and therefore, it is a mandatory declaration.
Trichinella spp. it is a complex of at least 12 taxa that presents a global distribution. To date, nine species and three genotypes not yet named are recognized within this genus. The taxa of Trichinella spp. are divided into two groups or clades according to their larvae encapsulate or not in the muscles of their hosts. The species clade with capsule infects only mammals, and includes T. spiralis (T1), T. native (T2), T. britovi (T3), T. murrelli (T5), T. nelsoni (T7), T patagoniensis (T12), and Trichinella genotypes T6, T8 and T9. The clade of uncapped species includes T. pseudospiralis (T4), which infects mammals and birds; T. papuae (T10) and T. zimbabwensis (T11), which infect mammals and reptiles. Most of the species have a wide geographic distribution and infect multiple hosts, only a few are located in specific areas and affect specific animals. More than 100 species of mammals, birds and reptiles have been identified as definitive hosts of this parasite. Most of the hosts are wild, constituting the main reservoir of Trichinella, especially those animals with great cannibalistic and scavenging activity. However, for human trichinosis, domestic hosts of the disease, such as pigs, horses, dogs, cats and rodents, are of particular importance.
It has been shown that eight of these taxa are infectious for humans, while the other four are considered potentially infectious. However, the main etiological agent of disease in humans is Trichinella spiralis. T. spiralis is the species best adapted to pigs (domestic and wild) and the most detected in them, also affecting equines and rats, among other animals. In addition, it has a cosmopolitan distribution since humans have introduced it in most of the continents passively through animals. Other species of relevance are the encapsulated T. native, for its wide geographical distribution, and T. pseudospiralis, which stands out among the non-encapsulated species as the only species that can infect both birds and mammals.
Morphologically, Trichinella is a dioecious nematode, filiform and small. So far, T. spiralis is the smallest known nematode that parasitizes humans. In the adult stage, the females are larger than the males, measuring 3-4 mm in length and about 60 μm in diameter, while the male measures 1.3-1.5 mm with about 40 μm in diameter. The males are thinner in the anterior part than in the posterior end, where they present two lobed caudal appendages, a copulatory pseudobursa. The infecting forms, L1 larvae in nurse cells, measure about 1.2 mm and about 35-40 μm in diameter.
The life cycle of Trichinella spiralis is an autoheteroxene cycle, since all the phases occur within a same host that fulfills a mixed role, acting as both intermediate and definitive host. In the life cycle of Trichinella there is no evolutionary form in the external environment, both the cycle and the life of this parasite are mainly intracellular (with the exception of migratory larvae), and it occurs in the intestinal microvilli and muscle tissue of the host. Transmission from one host to another, animal or human, occurs through the ingestion of muscle tissue with encysted Trichinella L1 larvae. Wild animals are infected by predation and ingestion of waste; domestic pigs for feeding with leftover meat, uncooked meat products, or for eating rats; and in man, by consumption of meat or insufficiently cooked meat products from an infected animal. The existence of two biological cycles is contemplated, the domestic cycle and the wild cycle, depending on the animals involved in the transmission. The main sources for human infection are domestic pigs and wild boars. In developed countries where trichinosis has been controlled in domestic pigs, human infection is most often due to the consumption of game or horse meat. However, in Europe, EFSA recognizes the sporadic presence of Trichinella in pigs, mainly those raised in the wild and for self-consumption. In addition, most cases in the world are still caused by the consumption of insufficiently cooked pork.
The cycle begins when a carnivore eats meat containing the viable encysted L1 larvae, the infective forms of Trichinella spiralis. Once in the small intestine of the host, the larvae are released from the cysts by the action of proteolytic enzymes and invade the intestinal mucosa, where they evolve into adults, intracellular parasites of a enteroepithelial syncytium. After the copulation, the females, larviparous, lay their larvae for weeks until the host's immune response affects their viability. The production of larvae by the female depends on the species of the host, the degree of infestation, the immune status of the host, and the species of Trichinella parasite. After about 6 weeks, the female is dragged with the food bolus and is eliminated with feces. Newly hatched larvae, released in the intestinal submucosa, migrate via blood and lymph to other organs or tissues of the host. These larvae have a specific tropism for striated muscle, and have a preference for muscles with high activity and low glycogen levels, such as the diaphragm, biceps, tongue and masseters. Also, they can invade any other type of tissue, but only the larvae that reach the host's striated musculature will mature and become L1 larvae. The larvae invade the muscle fibers, which are transformed into nurse cells, and in the sarcolemma they are encapsulated as L1 larvae, forming a cyst where the muscular larva remains alive for years.
Trichinella infection in animals usually occurs without clinical symptoms, even in animals that reach a high parasitic load. However, in man, trichinosis manifests as an acute toxic-infection, severe disease and unspecific, whose clinical picture varies depending on the infectious dose, the individual's susceptibility and possibly Trichinella species. The infection can be asymptomatic if it involves a low number of larvae. However, in the case of the ingestion of a few hundred larvae, after two days gastrointestinal symptoms will appear, followed by the development of a serious disease, although rarely fatal. The clinical signs of the disease usually last from 4 to 6 months, although occasionally a longer duration has been reported, up to 2 years. Symptomatological alterations are divided into three successive stages: the intestinal phase, migration phase, and state. In the intestinal phase, the clinical manifestations are due to the nematodes growing and invading the intestinal villi and they appear after a period of incubation that oscillates between 2 and 10 days after the ingestion of meat contaminated with larvae. The symptoms consist of abdominal pain, diarrhea, malaise, fatigue, hyporexia, nausea, vomiting and fever, which persist for 10 to 12 days. The systemic phase, migration and muscle invasion, manifests approximately 5 days later, although it can manifest itself in a period of between 1 and 6 weeks after infection. As the larvae migrate to muscles, causing acute pain, headache, high fever, periorbital and facial edema, photophobia, chills, cough, lacrimation, pain in joints and muscles, muscle stiffness, rashes, conjunctival hemorrhage and subungual, fatigue, weight loss, and diarrhea or constipation. Although the larvae can penetrate in a multitude of cells, those that damage with more seriousness are those of the cardiac muscle, brain and retina. If the affected muscle is the diaphragm, hiccups and pain occur when breathing. The most serious manifestation in this phase is myocarditis, which can lead to death. The invasion of larvae to the central nervous system causes headache, insomnia, irritability, apathy, vertigo, and meningeal data. Finally, in the state phase, the larvae have already established themselves in the muscle and the predominant symptom is muscle pain that is exacerbated by exercise. Over time, the symptoms disappear and the cystic wall becomes calcified.
Available laboratory tests
Diagnostic tests for the detection of Trichinella spp. are of vital importance to eliminate infected animals from the food chain and prevent disease in humans. In Europe, Regulation (EC) No. 2015/1375 establishes specific rules for official controls of the presence of Trichinella in meat. Diagnostic tests are grouped into two categories: direct and indirect methods.
The direct methods are based on the detection of larva, encysted or free, in striated muscle tissue. Trichinella larvae are usually located in higher concentrations in the preferred muscles, which vary according to the host species. It is important to sample these preferred muscles to maximize sensitivity. For example, the muscles of parasitological interest and those that concentrate the largest number of larvae of T. spiralis in the porcine species, are the diaphragm, tongue, masseter and intercostal, while, in feral pigs, the preferred muscles include the front leg, the tongue or the diaphragm; and in horses, the muscles of the tongue tend to lodge most of the parasites, followed by the masseter, the diaphragm and the muscles of the neck.
The compression or trichinoscopy method consists in the microscopic observation of the muscle sample placed between two tightly bound slides, in order to detect calcified cysts with larvae inside. Trichinoscopy is a less sensitive method and is not recommended as a reliable test for inspection of carcasses, neither in terms of food safety nor in terms of surveillance of infections. In addition, the trichinoscopic examination fails to detect non-encapsulated Trichinella species that infect domestic and wild animals and humans. Therefore, it is not considered an adequate detection method by European legislation (Regulation nº 2015/1375). However, due to its simplicity, it can be used as a complementary method.
Artificial digestion methods include the enzymatic digestion of individual or grouped muscle tissue samples, and use mechanical homogenization or grinding, shaking and incubation. Subsequently, by means of filtration and sedimentation procedures, all the larvae that have been released from the muscle during the digestion are recovered and concentrated. The samples processed with these methods are subjected to a stereomicroscopic examination to check the presence of larvae. Digestion methods are more sensitive than trichinoscopy, with them you can detect <1 larva per gram of tissue. The main limitations of the technique are the possible low levels of infection, the amount of muscle digested, the unequal distribution of larvae within tissues, and the poor digestibility of certain tissues and samples of wildlife that have been frozen. Therefore, if the sample cannot be extracted at the preferred site or if the type or species of animal has a higher risk of infection, Regulation (EC) No. 2015/1375 will increase the size of the samples for the parasitic analysis, such as a minimum of 3-5 g in the case of pigs and 5-10 g in the case of horses or hunting animals. This regulation indicates artificial digestion as a reference method for routine use for the analysis of individual and collective samples.
Indirect methods for the detection of Trichinella infection can be of different types, but serological tests are the most frequently used. The sensitivity and specificity of serological methods depend above all on the type and quality of the antigen used. Most of the data related to the realization and validation of serological tests come from its application to pigs. Current European legislation dictates that serological tests can be useful for surveillance if they have been validated by a reference EU laboratory, designated by the Commission. Serological tests are acceptable for monitoring or verification of Trichinella free herds. However, serological tests are not adequate to detect the presence of Trichinella in individual animals intended for human consumption.
Finally, molecular biology techniques for genotyping have confirmed the current taxonomic classification. In addition, different protocols for the detection of Trichinella in meat have been described by the specific amplification of their DNA by PCR. However, currently the use of this technique is mostly epidemiological. The different species and genotypes of the genus Trichinella spp. they are morphologically indistinguishable, and their identification is based on the use of biochemical or molecular assays. The isolation of Trichinella larvae from muscles of infected animals allows the identification of larvae at the species or genotype level in order to obtain valuable epidemiological information to control these zoonotic pathogens.
Tests offered by IVAMI:
- Test of direct microscopic examination (trichinoscopy), with examination of 5 fractions of 1 g for each meat piece received.
- Microscopic examination after pepsic digestion during 24 hours of individual piece of meat (30 g), or collective (up to 100 g of samples at a time).
- Identification of species of the genus Trichinella spp. (PCR and sequencing).
For samples of pig, muscle samples are recommended (in order of interest), diaphragm, tongue or base of tongue, right or left masseters, right or left intercostals, shoulder, leg, and spine. Depending on the type of sample and analysis there are different specifications:
- In the case of whole carcasses of domestic pigs, a sample of a minimum weight of 1 g should be taken on one of the pillars, of the diaphragm.
- In the case of breeding sows and boars, a larger sample of a minimum weight of 2 g should be taken on one of the pillars of the diaphragm, in the transition zone between the muscular part and the tendinous part.
- When the diaphragm abutment is not available, a double-sized sample, 2 g (or 4 g in the case of breeding sows and boars), should be taken from the part of the diaphragm located near the ribs or sternum, or of the masseters, the tongue or the abdominal muscles.
- For pieces of meat, a sample of a minimum weight of 5 g of striated muscle, which contains little fat and, if possible, is located close to the bones or the tendons.
- In the case of frozen samples, it will be taken to analyze a sample with a minimum weight of 5 g of striated muscle.
When wishing to analyze other animals, samples of at least 10 g of the front leg, tongue or diaphragm of the wild boar should be taken; muscles of the tongue or masseters of horses; samples of the diaphragm, the masseter muscle and the tongue in the bears; muscles of the tongue for the walrus; the masseter, pterygoid and intercostal muscles in crocodiles; and the muscles of the head in the case of birds.
Conditions of conservation and shipment of the samples
Samples should be stored refrigerated and shipped at least under conditions that guarantee refrigeration during transport. The sample must be placed in a biological safety container or flexible plastic bottle (polypropylene), and must be sent with refrigeration in a box of expanded polystyrene (white cork), with a cold storage. The sample, if possible, must not have been previously frozen. If it has frozen, notice it in the application.
Delivery of results:
- For trichinoscopy and artificial digestion of samples: 24 to 48 hours.
- For molecular identification at the species level: 4 to 5 days.
Cost of the tests:
- Cost per sample received for both methods: consult firstname.lastname@example.org.
- For samples received simultaneously in groups of 5 or more samples for both methods (each): consult email@example.com.
- For analysis of collective samples by the digestion method (up to 100 g); consult firstname.lastname@example.org.
- Molecular identification of the species of Trichinella spp.: consult email@example.com.