61 Trichinellosis

Trichinellosis is caused by nematodes of the genus Trichinella. These zoonotic parasites show a cosmopolitan distribution in all the continents, but Antarctica. They circulate in nature by synanthropic-domestic and sylvatic cycles. Today, eight species and four genotypes are recognized, all of which infect mammals, including humans, one species also infects birds, and two other species infect also reptiles.

Parasites of the genus Trichinella are unusual among the other nematodes in that the worm undergoes a complete developmental cycle, from larva to adult to larva, in the body of a single host, which has a profound influence on the epidemiology of trichinellosis. When the cycle is complete, the muscles of the infected animal contain a reservoir of larvae, capable of long-term survival. Humans and other hosts become infected by ingesting muscle tissues containing viable larvae.

The symptoms associated with trichinellosis vary with the severity of infection, i.e. the number of viable larvae ingested, and the time after infection. The capacity of the worm population to undergo massive multiplication in the body is a major determinant. Progression of disease follows the biological development of the parasite. Symptoms are associated first with the gastrointestinal tract, as the worms invade and establish in the small intestine, become more general as the body responds immunologically, and finally focus on the muscles as the larvae penetrate the muscle cells and develop there. Although Trichinella worms cause pathological changes directly by mechanical damage, most of the clinical features of trichinellosis are immunopathological in origin and can be related to the capacity of the parasite to induce allergic responses.

The main source of human infection is raw or under-cooked meat products from pig, wild boar, bear, walrus, and horses, but meat products from other animals have been implicated. In humans, the diagnosis of infection is made by immunological tests or by direct examination of muscle biopsies using microscopy or by recovery of larvae after artificial digestion. Treatment requires both the use of anthelmintic drugs to kill the parasite itself and symptomatic treatment to minimize inflammatory responses.

Both pre-slaughter prevention and post-slaughter control can be used to prevent Trichinella infections in animals. The first involves pig management control as well as continuous surveillance programmes. Meat inspection is a successful post-slaughter strategy. However, a continuous consumer education is of great importance in countries where meat inspection is not mandatory.

The unravelling of the nature of trichinellosis may reach back to antiquity as suggested by historical references to diseases that bear striking similarities to clinical aspects of Trichinella infection. The earliest such case involved a young Egyptian living along the Nile about 1200 B.C. (Gould 1970; Campbell 1983a). There is evidence of human infections even in prehistoric cultures (Owen et al. 2005). The modern history of trichinellosis, however, begins in 1835, with the discovery by microscopy of the larval stage of the parasite by James Paget and Richard Owen in London. It was Owen who coined its first name, Trichina spiralis (Owen 1835). The parasite was first found in animals by Leidy (1846), who identified larval cysts in muscles from a pig and realized that they were identical to those described in humans. Evidence that infection was acquired by ingestion of live larvae from infected muscles was obtained in the 1850s by feeding experiments using dogs and other animals (Virchow 1859). The first account of transmission to humans from infected pig meat, and association with a defined set of symptoms, was published by Zenker (1860) who made a detailed study of a patient who had been clinically diagnosed as having typhoid. Post-mortem examination of muscle tissue, however, confirmed the presence of a heavy infection of Trichinella, and the symptoms recorded before death (fatigue, fever, oedema, muscle and joint pain) are now recognized as characteristic of trichinellosis and largely immunopathological in origin. The patient had eaten infected ham and sausages shortly before the onset of her symptoms, and similar symptoms also occurred in other members of the household who had eaten these items. Zenker’s association of a defined pathogen with a defined disease was a milestone in medical microbiology, though it rarely receives the recognition it deserves, being overshadowed by later discoveries in bacteriology.

For 150 years after its first scientific description, Trichinella spiralis was considered the sole member of the genus, having a phenomenally wide host range (Campbell 1983b). However, beginning in the 1950s and 1960s, scientists began reporting an increasing number of host-specific behaviours among different geographic isolates (Rausch et al. 1956; Nelson et al. 1966). These studies, conducted over the next 30 years, yielded a remarkable series of new findings on the genetic diversity within the genus resulting in a new Trichinella taxonomy encompassing eight species (Table 61.1), along with a more complete zoogeographical and epidemiological knowledge base (Pozio et al. 1992; Pozio and Zarlenga 2005;www.iss.it/site/Trichinella/index.asp).

The genus Trichinella comprises a monophyletic lineage in the family Trichinellidae, the putative sister to the Trichuridae (Capillariinae, Trichurinae and Trichosomoidinae). The superfamily Trichinelloidea to which Trichinella belongs is phylogenetically diagnosed by the stichosome, a region of the glandular oesophagus, and the bacillary bands, an assembly of structural characters unknown among the other nematodes. Today, two main clades are recognized in the genus Trichinella; one that encompasses species that encapsulate in host muscle tissue, and a second that does not encapsulate after muscle cell dedifferentiation (Pozio and Murrell 2006).

Five species and four genotypes of undetermined taxonomic status infecting only mammals belong to this clade (Table 61.1).

Trichinella spiralis sensu stricto (Owen 1835) is the first species discovered and the most characterized because of its importance both as a cause of human disease and as a model for basic biological research investigations, due in large part to its relatively high frequency in both domestic and sylvatic animals and to its high infectivity for laboratory animals. Dissemination of the parasite and its hosts was especially facilitated by the European colonization of North, Central and South America, New Zealand, Hawaii, and Egypt from the sixteenth to twentieth centuries. In many regions of the world this species has been transmitted to wildlife hosts through exposure to garbage dumps or foraging near human settlements, where pork scraps and offal from slaughtered animals were scattered in the environment (Pozio and Murrell 2006). At the world level, most of human infections are due to this species.

Trichinella nativa (Britov and Boev 1972) is usually named as the arctic or freeze resistant species and is widespread among wild carnivores of the arctic and subarctic areas of America, Europe and Asia. The main biological features of T. nativa are a very low infectivity to swine and a high resistance to freezing in muscles of carnivores (Pozio and Zarlenga 2005). Human populations living in frigid zones acquire T. nativa infection by eating raw meat from walruses, bears and other game animals. The genotype, Trichinella T6, phylogenetically related to T. nativa, is widespread in carnivores of Canada and Alaska, and along the Rocky Mountains and Appalachians in the USA. Sporadic infections in humans have been documented for the consumption of game meat.

Trichinella britovi (Pozio et al. 1992), is a Palearctic and African species. European and Asian isolates of this species were previously named T. nelsoni by Russian scientists (Pozio et al. 1992). Among sylvatic species, T. britovi has the widest geographical range, occurring in wild carnivores and wild boars of the temperate areas of Europe and Asia, and extending southward to northern and western Africa (Pozio et al. 2005a). This parasite can reach domestic pigs from extensive grazing systems or feed with scraps from sylvatic carnivores (Pozio and Zarlenga 2005). A percentage of human infections occurring in Europe, Asia, North and West Africa are caused by this species. The genotype Trichinella T8, very similar to T. britovi, has been identified in wild animals of South Africa and Namibia. No human case due to this genotype has been documented.

Trichinella murrelli (Pozio and La Rosa 2000), is spread among sylvatic carnivores across the USA and in some southern regions of Canada. This species does not develop in swine. It is the causative agent of infection in humans especially from consumption of meat from black bears. A great deal of clinical information on this species was gained from a 1985 outbreak in France due to the consumption of horse meat imported from USA (Ancelle 1998). Trichinella isolates from Japanese wildlife, originally identified as T. britovi, are now designated as a separate genotype, named Trichinella T9, which is phylogenetically related to T. murrelli (Pozio and Zarlenga 2005; Zarlenga et al. 2006).

Trichinella nelsoni (Britov and Boev 1972), sensu stricto (Pozio et al. 1992), has been detected in eastern Africa, from Kenya to South Africa. The host range includes sylvatic carnivores and, at least occasionally, bush pigs and warthogs, some of which have been the source of infection for humans. Less than 100 human infections have been documented for this species in Kenya and Tanzania (Pozio 2007).

Trichinella T12 is a new encapsulated species of Trichinella, recently detected in mountain lions (Puma concolor) from Patagonia, Río Negro, Argentina. The available information is the molecular structure of two non-coding and one coding sequences that are different from those of the eleven currently recognized species/genotypes of the genus Trichinella (Pozio et al. 2009).

One species infecting mammals and birds, and two species infecting mammals and reptiles, compose this clade (Table 61.1). The main biological features of these parasites in comparison to those of the previous clade are the lack of a collagen capsule and their infectivity to other vertebrates in addition to mammals.

Trichinella pseudospiralis (Garkavi 1972) is a cosmopolitan species infecting both mammals and birds. Three populations, which can be distinguished on a molecular basis, have been detected in the Palaearctic, Nearctic, and Australian (Tasmania) regions (La Rosa et al. 2001). This parasite has been found in 14 mammalian species including domestic and sylvatic swine, and 13 avian species (Pozio 2005), where the number of reports in mammals is much higher than that in birds. Infections in humans, with some deaths, have been documented in Kamchatka, Thailand, and France (Pozio and Murrell 2006).

Trichinella papuae (Pozio et al. 1999), circulates in both mammals and reptiles (domestic sows, wild pigs, and farmed saltwater crocodiles) of Papua New Guinea and Thailand (Pozio et al. 2005b; Pozio 2007). Infections in humans have been documented (Owen et al. 2005; Kusolsuk et al. 2010).

Trichinella zimbabwensis (Pozio et al. 2002), very similar to T. papuae, has been detected in wild and farmed reptiles of Africa (Zimbabwe, Mozambique, South Africa, and Ethiopia), in experimentally infected mammals (Pozio and Murrell 2006) and in a lion. Human infections have yet to be reported.

The phylogeny of species and genotypes of the genus Trichinella shows that the extant species of Trichinella diversified within the last 10 to 20 million years, which coincided with the divergence of Suidae from the Tayassuidae in the Lower Miocene in Eastern Asia. Trichinella spiralis appears to be the oldest of the encapsulated clade (Zarlenga et al. 2006; Pozio et al. 2009). The switch of non-encapsulated species to birds or reptiles occurred more recently. The introduction of T. nelsoni, Trichinella T8, and T. britovi into Africa is the result of three independent expansion events from Eurasia following the land connections that formed during upper Miocene and into the Pleistocene (Pozio et al. 2005a). According to Zarlenga et al. (2006), ursids, canids and felids are principally responsible for the radiation of Holarctic species throughout Europe and into North America through Beringia.

A peculiarity of the cycle of nematodes of the genus Trichinella is the development of two generations in the same host (Fig. 61.1). Larvae are released from the cysts by digestion in the stomach and pass into the small intestine, where they penetrate rapidly into the epithelial cells of the mucosa, occupying an intracellular niche. At this stage the larvae are about 1 mm long occupying some 40–50 enterocytes. Subsequent development is extremely rapid; the parasite undergoes four moults in about 30 hours to reach the immature adult stage and increases in length 2–3 times. As with all nematodes, the sexes are separate, and the female is the larger, reaching 3 mm in length.

Males and females mate in the small intestine, the eggs of the female are fertilized, develop rapidly in the uterus, and the female then begins lo release newborn larvae (NBL, the first larval stage) directly into the mucosa. Release continues for several days or possibly weeks, and is eventually terminated by the onset of immunity or by senility of the worm. The number of NBL produced during the lifetime of each female is dependent on many factors, and differs between the various species (100–1,500 per female). The severity of trichinellosis as a disease results largely from this capacity of the worm population to undergo massive multiplication in the body of the host.

After release from the female, NBL migrate into mucosal lymphatics, pass through the draining lymph nodes, enter blood vessels, and are carried around the body. Although they can complete their development only in striated skeletal muscles, larvae may attempt to penetrate other tissues, including the brain, heart, and kidneys. After penetration into striated muscle fibres the NBL lies free in the cytoplasm and induces a complex series of changes which result in the host cell becoming transformed into a quite different structure, the nurse cell that serves to ensure the growth, development, and survival of the parasite. With time the nurse cell becomes surrounded by a capsule of collagen and a network of capillaries to form the characteristic cyst. After 3–4 weeks the larva has grown considerably (from 100 µm to 1 mm), is resistant to digestion, and is capable of infecting another host to initiate a new infection cycle. Larvae can remain infective for very long periods (up to 20 years in bears and up to 40 years in humans); however, in some hosts with time, the cysts become calcified and the larvae die.

An important adaptation of the parasite, which facilitates its transmission, is the physiological mechanism utilized by muscle larvae to promote their survival in decaying carcasses. The greater the persistence of larval viability, the higher the probability to induce infection when ingested by a scavenging host. In spite of the larva-induced angiogenic process that develops around the nurse cell after larval penetration of the muscle cell, larval metabolism is basically anaerobic (Despommier 1990), which favours its survival in decaying tissues. The persistence of larvae in putrefying flesh is, of course, also determined by the environment: high humidity and low temperatures favour survival of encapsulated larvae even when the muscle tissue is completely liquefied. This condition has been proposed as the environment of the ‘free-living’ stage, resembling the egg stage of most of other nematode species. The importance of this stage in the natural cycle of the parasite is underscored by the survival of muscle larvae in frozen muscles of carrion for one (T. britovi) or more years (T. nativa and Trichinella T6) (Pozio and Murrell 2006). It is important to stress that the survival of muscle larvae to freezing occurs mainly when these larvae parasitize striated muscles of carnivores, whereas the survival time to freezing is strongly reduced to a few days or weeks when muscle larvae of the same strain parasitize other mammalian hosts such as swine or rodents.

Although T. spiralis was first discovered in domestic animals, the other species of this genus are primarily parasites of wildlife. When humans fail in the proper management of domestic animals and wildlife, Trichinella species are transmitted from the sylvatic environment to the domestic one, sometimes through synanthropic animals. In addition, they can transfer in a reversible path from domestic animals to wildlife.

The sylvatic cycle occurs in all continents with the exception of Antarctica, where there is neither a record of this nematode nor evidence of any searches for it in marine mammals and birds. Trichinella infections in wildlife have been documented in 66 (33.3%) countries of the world (Pozio 2007). These parasites have been reported in more than 100 species of mammals belonging to 11 orders, i.e. Marsupialia, Insectivora, Edentata, Primates, Lagomorpha, Rodentia, Cetacea, Carnivora, Perissodactyla, Artiodactyla, and Tylopoda (Pozio 2005). The transmission cycles of the different sylvatic species and genotypes are closely related to their host species ecologies. Among primates, only humans have been naturally infected with Trichinella. In spite of the potential broad host spectrum of Trichinella spp., the greatest biomass of these parasites occurs amongst the Carnivora and the artiodactylid family Suidae. The role of small mammals (mainly rodents and insectivores) in the sylvatic cycle is still uncertain due to the low number of infections in their populations and the lack of sufficient surveys on a large number of these mammals (Pozio and Zarlenga 2005). Trichinella nativa is commonly found in polar bears, and increasingly in walruses where it presents a significant zoonotic hazard. This has resulted in the implementation of food safety programmes in some arctic communities to test harvested walrus meat for Trichinella larvae prior to consumption (Proulx et al. 2002). Trichinella sp. has been reported very rarely in seals from the Arctic.

Seven species of birds are documented as hosts for T. pseudospiralis, and six other species suspected, but unconfirmed (Pozio 2005). Only three species of reptiles have been detected naturally infected in Africa (Nile crocodile and Nile monitor lizard) and in Papua New Guinea (saltwater crocodile). In addition, meat from a clouded monitor and a turtle has been implicated as the sources of infection of human trichinellosis outbreaks, which occurred in Thailand (Kambounruang 1991). A soft-shelled turtle has been implicated as a source of trichinellosis in Taiwan (Lo et al. 2009). There is a single report of an experimental infection of amphibians (frogs and axolotls) with T. spiralis, in which it was observed that the development of larvae in the muscles was incomplete. Attempts to infect fish with T. spiralis, T. britovi, T. pseudospiralis, T. papuae and T. zimbabwensis have also failed (Pozio and La Rosa 2005). The role of invertebrates as paratenic hosts of Trichinella species has been investigated in adult and larval stages of several insects and amphipods. The survival of Trichinella larvae in these paratenic hosts is under the influence of the environmental temperature, lower the temperature, longer the survival. The sylvatic cycle may be influenced by human actions such as the common habit of hunters of leaving animal carcasses in the field after skinning, or removing and discarding the entrails, which increases the probability of transmission to new hosts (Pozio et al. 2001).

This cycle has been documented in 43 (21.9%) countries of the world and it occurs where there are high risks farming practices such as the feeding of food waste containing pork scraps, or exposure to carcasses of dead swine, or wildlife (Gamble et al. 2000; Pozio and Murrell 2006; Pozio 2007). These risks are usually encountered in:

The most common etiological agent of the domestic cycle is T. spiralis, which is well adapted to swine and synanthropic hosts, in which it exhibits a very high reproductive rate without inducing serious pathology. Occasionally, T. britovi and T. pseudospiralis have been transmitted in the domestic cycle, when humans feed pigs with game meat scraps or ‘pasture’ pigs in refuse dumps containing carcasses of sylvatic animals. Also T. papuae and T. zimbabwensis are transmitted among farmed crocodiles fed with scarps from slaughtered crocodiles (Pozio and Murrell 2006).

In the domestic habitat, the brown rat (Rattus norvegicus) is frequently found to be infected with T. spiralis and infrequently with T. britovi or T. pseudospiralis (Pozio and Zarlenga 2005). In the nineteenth century, Leuckart proposed a ‘Rat Theory’, which implicated rats as a major reservoir of T. spiralis for domestic pigs. In 1871, Zenker suggested that the infection in rats was merely an indicator of Trichinella exposure risk in the area and that the real source of infection for both pigs and rats was meat scraps and offal of infected pig carcasses. This is consistent with findings that the occurrence of T. spiralis in domestic pigs greatly decreased when feeding with uncooked garbage and offal was terminated (Pozio and Zarlenga 2005).

Between 1975 and 2005, human outbreaks of trichinellosis have occurred in France (2,296 infected people in eight outbreaks) and Italy (1,038 infected people in seven outbreaks), from the consumption of meat from horses imported from Canada, the Former Yugoslavia, Mexico, Poland, and the USA (Pozio and Zarlenga 2005). In addition infections in 21 horses, bred in the Former Yugoslavia, Mexico, Poland and Romania, have been detected at the slaughterhouses (Pozio and Zarlenga 2005). Worldwide, only 35 infections (horses that were the source of infection for human outbreaks and positive horses detected at the slaughterhouse) have been documented in horses since 1975, with a prevalence of infection of about one infected horse per 250 thousand slaughtered horses (Pozio 2001a; Murrell et al. 2004). Horse-meat outbreaks have important consequences for public health because of the high number of infected persons resulting from consumption of meat from a single horse. This has a high impact in terms of medical costs, horse-meat market economics, which collapses after each outbreak, and in legal and administrative terms related to the implementation of control measures at the national and international level (Ancelle 1998).

Trichinellosis is the proper term for the human zoonotic disease also known as trichinosis or trichiniasis. Of 198 countries present in the world, 40 (20%) are small islands or city-states, where Trichinella sp. infections cannot develop for the lack of potential reservoirs. According to published data, clinical trichinellosis has been documented in people of 55 countries (27.8%) (Pozio 2007). On the basis of reports from these 55 countries, the yearly incidence of clinical trichinellosis has been estimated to 10,000 cases with 0.2% of deaths but the real figure of infections and contacts between these parasites and the human beings is rather unknown.

Trichinella infections in humans are more related to cultural food practices which include dishes based on raw or undercooked meat of different animal origins than to the presence of the parasite in the domestic and wild animals of the country. In France and in Italy, most of trichinellosis cases are due to the consumption of raw horse meat, because this food habit is strongly related to the French culture imported also in Italy (Boireau et al. 2000; Pozio 2001a).

In Finland, where there is a high prevalence of infection in animals, no infection leading to disease has been documented in humans, due to the practice of eating only well cooked meat (Pozio 2007). In Romania, the highest prevalence of trichinellosis in humans occurs in the Transylvanian region which was colonized by German people who have kept their food habits which are known to be risk factors for trichinellosis (Blaga et al. 2007). In Israel, Lebanon, and Syria, human outbreaks of trichinellosis have been documented following consumption of pork from wild boars only among the Christian populations or immigrants from Thailand (Pozio 2007). In Algeria and Senegal, where the majority of the human population is Muslim, trichinellosis has only been documented in expatriates from France and very seldom in the population (Gretillat and Vassiliades 1967; Nezri et al. 2006; Pozio 2007).

In most African countries south of the Sahara, human infection is seldomly documented in spite of the presence of Trichinella-infected wildlife, because about a third of all African populations are of the Bantu ethnic group, which rarely consumes meat and when they consume meat it is well cooked (Pozio 2007). In Indonesia, where most people are Muslims and do not eat pork, the island of Bali is one of the few areas of the country where the majority of people are Hindu and foreign tourists visiting this island acquired the infection (de Carneri and Di Matteo 1989). In South America, trichinellosis has been documented only in Argentina and Chile where a high percentage of the population consumes raw pork and pork products (Schenone et al. 2002; Ribicich et al. 2005).

Overall, the most important source of Trichinella infection for humans remains pork and its related products from domestic pigs. Important foci of human trichinellosis from pork occur in Central (Mexico) and South America (Argentina and Chile) (Ortega Pierres et al. 2000; Ribicich et al. 2005), in Asia (China, Laos, Myanmar, Thailand) (Takahashi et al. 2000; Pozio 2001b; Liu and Boireau 2002; Wang et al. 2006; 2007; Barennes et al. 2008) and Europe (Bosnia-Herzegovina, Bulgaria, Byelorussia, Croatia, Georgia, Latvia, Lithuania, Poland, Romania, Russia, Serbia, and Ukraine) (Pozio and Murrell 2006; Pozio 2007). The migration of persons from eastern to western countries of Europe, has resulted in several human outbreaks of trichinellosis in Denmark, Germany, Italy, Spain and the UK (Pozio and Marucci 2003; Gallardo et al. 2007; Stensvold et al. 2007). The increasing number of international travelers has resulted in many reports of tourists who acquired Trichinella infections for the consumption of pork from warthogs in Africa, of bear meat in Canada and Greenland, of pork from domestic pigs in China, Egypt, Indonesia (Bali Island), Laos and Malaysia, and wild boar meat in Turkey and Algeria (Pozio and Murrell 2006).

The parasitic cycle can be divided into two phases: an intestinal (or enteric) phase and a muscular (or parenteral or systemic) phase, which can co-exist for a period lasting from a few days to weeks (Fig. 61.1). This cycle occurs in all mammals, including humans, in birds, and in reptiles. The larval penetration of the intestinal mucosa causes modifications in the brush border of villi, the lamina propria, and the smooth muscles of the jejunum. The migration of newborn larvae in the different organs provokes an immediate reaction, which causes immunological, pathological, and metabolic disturbances and the various clinical phenomena observed during the acute stage of the infection (Murrell and Bruschi 1994; Capo and Despommier 1996; Kociecka 2000). The immunological reaction consists of the production of inflammatory cells (i.e. mast cells, eosinophils, monocytes, and T and B lymphocytes), of cytokines, and antibodies.

The penetration and development of larvae in the muscle cells cause the acquisition by the cell of a new phenotype called ‘nurse cell’, accompanied by the disappearance of sarcomere myofibrils, the encapsulation of the larvae (in the case of encapsulated species), and the development of a capillary network surrounding the infected cell (Capo et al. 1998). In addition, the sarcoplasm becomes basophilic, the cell nucleus is displaced to the centre of the cell, and the nucleoli increase in both number and size. The cell becomes more permeable, resulting in an increased release of muscle enzymes. The parenteral or muscular phase is associated with inflammatory and allergic responses caused by invasion of the skeletal muscle cells by the migrating larvae.

This invasion can directly damage the muscle cells, or indirectly stimulate the infiltration of inflammatory cells. A correlation between the eosinophil levels and serum muscle enzymes such as lactate dehydrogenase (LDH) and creatine phosphokinase (CK) has been observed in people with trichinellosis, suggesting that muscle damage may be mediated indirectly by these activated granulocytes (Ferraccioli et al. 1988).

The presence of larvae in the central nervous system causes vasculitis and perivasculitis, with diffuse or focal lesions. The NBL tend to wander, causing tissue damage before re-entering the bloodstream, or remain trapped and destroyed by the following granulomatous reaction. Neural cells may also be damaged by eosinophil degranulation products such as eosinophil-derived neurotoxin and major basic proteins (Durack et al. 1979).

Moreover, heart and brain lesions are often associated and could result from the simultaneous intervention of local prothrombotic effects of eosinophil activation and vascular injury caused by the migrating larvae (Fourestié et al. 1993). Myocarditis is triggered initially by invasion of the migrating larvae, then from immunopathologic processes such as activated eosinophil infiltration and mast cell degranulation (Paolocci et al. 1998). The length of survival of the nurse-cell parasite complex in the host is known to vary greatly from one to two years to an undetermined number of years, although survival for up to 30 years has been reported in humans (Fröscher et al. 1988).

In the early stage of the infection, the most common intestinal sign and symptom are diarrhoea and abdominal pain. This symptomatology usually precedes fever and myalgia by three to four days, and then disappears in less than one week. It has been observed that the shorter the duration between infection and the appearance of diarrhoea and fever, the longer the duration of both fever and facial oedema (Dupouy-Camet et al. 2002).

In most persons, the acute stage begins with the sudden appearance of general discomfort and severe headaches, an increase in fever, chills and excessive sweating. The major syndrome of the acute stage consists of persistent fever, periorbital or facial oedema, muscle pain, and severe asthenia, lasting for several weeks. Transient dizziness and nausea can also occur. Though less common, diarrhoea and conjunctival and sub-lingual haemorrhages are also observed. This is the stage during which the adults and the migrating larvae provoke the signs and symptoms of the disease. Fever is one of the earliest and most common sign of trichinellosis. Body temperature increases rapidly up to 39–40°C, and fever lasts from eight to ten days, although it can persist for up to three weeks when the disease is severe.

Oedemas are very typical signs of trichinellosis, although their intensity varies depending upon the intensity of the reaction to the infection. In the severe form of trichinellosis, oedema extends to the upper and lower extremities. The oedema is symmetrical. It usually vanishes rapidly following treatment with glucocorticosteroids. Muscle pain affects various muscle groups (cervix, trunk, masseters, and upper and lower extremities), and its intensity is related to the severity of the disease. The pain usually appears upon exertion, although most persons with severe trichinellosis or phlebitis associated with trichinellosis also experience myalgia at rest. Some persons with severe disease become disabled with a muscle weakness. The restriction of movement due to the pain associated with exertion leads to contractures of the upper and lower limbs, nuchal pseudorigidity, and trismus. Severe myalgia generally lasts 2–3 weeks.

Eosinophilia has been observed in most case of trichinellosis, with few exceptions. It appears early, before the development of clinical signs and symptoms, and it increases between the 2–5 weeks after infection. Eosinophilia can be low (< 1,000/mm³), moderate (1,000–3,000/mm³), or high up to 19,000 cells per mm³. It regresses slowly and can remain at lower levels for a period of several weeks to three months. The level of eosinophilia is correlated with the degree of myalgia and is higher in persons with neurological complications (Ferraccioli et al. 1988; Fourestié et al. 1993). The levels of muscle enzymes (CK, LDH, aldolase, and aspartate aminotransferase) in serum increase in 75–90% of infected persons between 2–5 weeks after the infection. In the course of trichinellosis, there is an increase in total IgE. Clinical observations suggest that Trichinella-specific IgE are responsible for allergic manifestations typical of the clinical picture of trichinellosis, such as cutaneous rash or oedemas (Watanabe et al. 2005).

Complications usually develop within the first two weeks in severe cases, but they have also been reported in moderate cases, in persons who were improperly treated (including those for whom treatment was begun too late) and, particularly, in the elderly. A positive correlation has been reported between age and the frequency and severity of complications (Dupouy-Camet et al. 2002). Encephalitis and myocarditis, which are both life-threatening, are often simultaneously present (Fourestié et al. 1993). Cardiovascular disturbances can occur in moderate or severe cases of trichinellosis, usually between the third and fourth week p.i.) (Bessoudo et al. 1981; Compton et al. 1993, Lazarevic et al. 1989; Puljz et al. 2005). Myocarditis develops in 5–20-% of all infected persons. The symptoms include pain in the heart region, tachycardia, and electrocardiogram abnormalities. Neurological complications include a variety of signs and symptoms (Fourestié et al. 1993). Neurological complications could be less frequent if the infected person is treated early. Ocular lesions appear during the acute stage of the disease and result from disturbances in microcirculation (Pozio et al. 2003). Dyspnea is relatively common and is caused primarily by parasite invasion and subsequent inflammation of respiratory muscles such as the diaphragm. Digestive complications occur during the acute stage of infection, and they consist of massive protein exudation leading to hypoalbuminemia and localized oedemas, acute intestinal necrosis, and prolonged diarrhoea (Pozio et al. 2003).

The severity of the disease depends on a number of variables which are often interrelated, including the number of larvae ingested, the frequency of consumption of infected meat; how the meat was cooked or treated (e.g. whether it was raw or rare or whether it had been smoked or salted); the amount of alcohol consumed at the time of meat consumption, given that alcohol could increase the resistance to the infection (Pawlowski 1983); the Trichinella species involved (the number of NBL shed by females differs by species); and individual susceptibility which depends on ethnic factors as well as sex, age, and the immune status of the host. The length of the incubation period depends upon the same variables as disease severity. Furthermore, it has been observed that for the more severe forms of trichinellosis, the incubation period is generally shorter, specifically: the incubation period lasts approximately one week for the severe form, two weeks for the moderately severe form, and at least three to four weeks for the benign and abortive forms.

Death is rare. Of the more than 6,500 infections reported in the EU in the past 25 years, only five deaths have been observed, all of which were due to thromboembolic disease, in persons over 65 years of age; deaths has been reported in two outbreaks involving more than one thousand cases (Ancelle et al. 1988). Forty-two fatalities out of 70,987 cases were reported in a worldwide survey between 1986 and 2009. No death was reported in outbreaks caused by T. britovi.

The convalescent stage of trichinellosis begins when the adult females cease to release migrating larvae and the already established larvae have completed their development in the muscle cells. The transition to this stage is characterized by the progressive disappearance of the signs and symptoms of the disease and by the return of laboratory parameters to normal values. This stage usually begins between the sixth and the eight week p.i., and infected persons could still have a severe asthenia for several weeks and chronic muscular pain for up to six months. Most persons will then become asymptomatic, though live larvae will persist in their muscles for years.

Whether or not a chronic form of trichinellosis actually exists is still under debate, and chronic trichinellosis could be difficult to distinguish from sequelae of the acute phase. However, its existence is supported by reports of persons who complain of chronic pain and a feeling of general discomfort and who show signs of paranoia and a syndrome of persecution, months or even years after the acute stage. The existence of a chronic form is supported by the presence of IgG antibodies in the serum, of bioelectric muscle disturbances, and of inflammatory cells in the muscles, all due to the chronic presence of live larvae. Moreover, this syndrome can also result from unnoticed brain localizations during the acute phase of the disease (Dupouy-Camet et al. 2002).

Although it is certain that Trichinella cause pathological changes as a result of mechanical damage to the intestine and to muscle tissues, the majority of the clinical features of trichinellosis are immuno-pathological in origin and can be related to the capacity of Trichinella to induce allergic responses, a property shared by many helminth species. The molecular basis is still undefined, although clearly it must reflect particular characteristics of worm antigens and the manner in which they are presented to the host. Studies in mouse models show that Trichinella infections preferentially stimulate cells of the T helper 2 (Th2) subset of CD4+T lymphocytes (Grencis et al. 1991), which release the cytokines necessary for the development of many of the allergic components of the disease, and it can reasonably be assumed that a similar situation exists during human infections.

During the intestinal phase, there is a marked infiltration of inflammatory cells, including neutrophils, eosinophils, and mast cells into the gut mucosa. Significant changes take place in mucosal architecture (e.g. villous atrophy), fluid flux across the mucosa is disturbed, mucus production is increased, and intestinal transit time is decreased. All of these changes are the result of T-cell activity and all can be related to the symptoms appearing during the intestinal phase of infection, of which diarrhoea is the most characteristic. Eosinophilia is a consequence of T-cell responses to both the intestinal adult worms and the muscle larvae and is dependent upon release of the cytokine IL-5 (Herndon and Kayes 1992). It has been shown in rodents that infection stimulates both parasite-specific IgE and total IgE antibodies as well as IgG isotypes (IgGl) that are involved in hypersensitivity reactions (e.g. Gabriel and Justus 1979) and this is consistent with the dominance of the T-cell response by the Th2 subset. Trichinella-specific IgE responses have been detected in humans (Bruschi et al. 1990).

The diagnosis of trichinellosis should be based on the anamnesis (source of infection, amount of infected meat ingested, number of larvae present in the infected meat, and number of cases in the epidemic focus), clinical evaluation (recognition of the signs and symptoms of trichinellosis and definition of the form of the disease, which significantly affects the choice of treatment), and laboratory tests (immunodiagnosis and/or detection of larvae in a muscle biopsy). An algorithm which can be used for the diagnosis is shown in Table 61.2. The most highly recommended technique is ELISA with excretory/secretory antigens which have the highest ratio between sensitivity and specificity and is best used in combination with immunoblotting to confirm ELISA-positive samples. In regions with the circulation of other parasite infections, a high number of false positive reactions can be detected (Gomez Morales et al. 2008). Seroconversion occurs between 12 and 60 days post infection. For parasitological diagnosis, a muscle biopsy must be collected, preferably from the deltoid muscle, although any skeletal muscle could be used. The surgeon should carefully collect 0.2–0.5 g of muscle tissue (less than a pea size) without fat or skin. The sensitivity of the parasitological diagnosis depends on the amount of muscle sample tested and the number of larvae per gram. Larvae in the muscle biopsy can be detected by three methods.

In adults, mebendazole should be administered at a daily dose of 25 mg per kg body weight (administered in 2–3 doses) for 15 days. The efficacy of mebendazole against larvae in muscle tissues depends on the time between infection and treatment and could be dose-dependent. Albendazole should be used at a daily dose of 800 mg/day (15–20 mg/kg/day) administered in 2–3 doses, for 15 days; in children over two years of age, the drug is given at 10 mg per kg body weight. For severe infection, the treatment may be repeated after five days. Glucocorticosteroids are used by most physicians to treat the signs and symptoms. The most commonly-used glucocorticosteroid is prednisolone, which should be administered at 30–60 mg/day in multiple doses until the symptoms and signs disappear. They must always be used in combination with anthelmintics and never alone, since they could increase the larval burden by delaying the intestinal worm expulsion.

The diagnosis of Trichinella infection in animals falls into two categories. Direct methods consist of identification and visualization of the first-stage muscle larvae encysted or free in striated muscle tissue. Indirect methods consist of detection of specific circulating antibodies.

The identification of Trichinella larvae in muscle samples is limited to post-mortem inspection of carcasses. In many countries, in order to prevent human trichinellosis, the examination of muscle samples from pigs and other susceptible animal species used for consumption (e.g. horses, wild boars, bears), should be a part of routine slaughter inspection (Gamble et al. 2000). Direct detection is also widely applied in wildlife epidemiology, where indicator animals (e.g. foxes, raccoon dogs, wild boars) are examined to assess the existence of infection among wildlife reservoirs. The sensitivity is greatly influenced by the muscle selected for sampling and the specific method used (Nöckler et al. 2000). Selection of muscles for sampling in meat inspection requires identification of predilection sites in a particular animal species. For routine meat inspection, it is necessary to ensure a sensitivity of approximately 1–3 larvae per g (lpg) as this is the level above which infection constitutes a food safety issue. Using the pooled sample digestion method, a minimum of a 1 g sample of tissue from a predilection site is examined. Using trichinoscopy, the examination of 28 oat kernel-size pieces of diaphragm muscle which corresponds to a 0.5 g sample, is recommended (Gamble et al. 2000). To ensure a high sensitivity in horse meat, 5 g samples should be examined by the pooled sample digestion method. For epidemiological studies in reservoir animals (wildlife), the sample size should also be adjusted upward to achieve a sensitivity of less than 1 lpg. Predilection muscles in carnivores may require a prolonged digestion time (up to 2 hours) (Kapel et al. 2005).

At least four artificial digestion methods are available:

The magnetic stirrer method is considered the gold standard because it is a method specifically designed for pooled samples and it has been subjected to validation studies (Kapel et al. 2005). The International Commission on Trichinellosis (www.med.unipi.it/ict/welcome.htm) recommends that all slaughter testing methods for Trichinella detection in pigs, other livestock and game should be validated by standard procedures and any new method be subjected for evaluation by at least three reference laboratories (Gamble et al. 2000).

Serological methods are used either for ante-mortem or post-mortem examination of serum samples for Trichinella-specific antibodies (World Organization for Animal Health (OIE) 2004). Serological methods are not recommended as a substitute for meat inspection of individual carcasses (Gamble et al. 2000). However, serological methods for detection of Trichinella infection are considered to be suitable for surveillance and epidemiological investigations in animal populations, where the prevalence of infection is high (Gamble et al. 2004). The ELISA method, the most commonly used method for the detection of Trichinella infection, provides an acceptable balance of sensitivity and specificity (OIE 2004). In many experimental and/or field studies, the successful use of an indirect ELISA for the detection of specific Trichinella antibodies in pig serum and meat juice samples has been demonstrated (Murrell et al. 1986; Nöckler et al. 2004).

The early stage of Trichinella infection is characterized by a ‘diagnostic window’ in which larvae have become encysted in muscle tissue as early as 17 d.p.i., but specific antibodies can not yet be detected in the host animal. In this case, false-negative results may occur when compared with direct tests (OIE 2004). Under normal conditions, serum antibodies decline slowly after an initial peak. In horses, specific antibody titres fell below cut-off levels of the ELISA as soon as 14 weeks p.i. (Soulé et al. 1989, 1993), and in naturally infected horses, specific circulating antibodies were not detected in spite of the presence of a high worm burden in muscles (Pozio et al. 1997, 1999). Considering the present state of knowledge, serological methods cannot be recommended to detect anti-Trichinella antibodies in this host (Gamble et al. 2004).

In contrast to control measures, prevention of pig infection with Trichinella has received substantially less attention, and most gains in reducing infection in domestic pigs have been the by-product of other disease prevention initiatives. For example, the introduction of garbage cooking laws in the USA was intended to control vesicular exanthema and hog cholera (Zimmerman and Zinter 1971; Zimmerman et al. 1973). Likewise, improvements in swine husbandry, including the introduction of confinement housing systems, generally occurred without any intention to prevent exposure of pigs to Trichinella. Despite an overall reduction in the prevalence of Trichinella infection in domestic pigs in some countries, resulting from a transition to confined management systems and improved veterinary public health efforts, the increase in prevalence rates in other countries where organized farming systems have broken down, underscores the ongoing risk of infection with this parasite in domestic pigs. Although most pigs are produced in confinement, the production of free-ranging pigs has increased in many countries, and obviously such pigs have a higher risk of exposure.

Recently, in some eastern European countries, the breakdown of organized farming systems and a decline in the availability and quality of veterinary services have resulted in higher prevalence rates in pigs and outbreaks of trichinellosis in humans (Djordjevic et al. 2003; Cuperlovic et al. 2005; Blaga et al. 2007).

The risk of Trichinella infection for pigs raised in outdoor farming systems is clear in all parts of the world (Gamble et al. 1999; Liu and Boireau 2002; Nöckler et al. 2004; Ribicich et al. 2005). Nevertheless, the degree of risk to pigs raised outdoors depends in great part on the infection level in local wildlife, and this degree of risk is of substantial importance for ‘organic’ or ‘green’ pig producers, who provide products to consumers seeking meat from animals raised under natural conditions. The so-called ‘backyard pigs’ are often fed food scraps or other forms of meat-containing waste and have ready access to rodents and wildlife. To compound the problems, pigs raised in this manner are generally not sold through retail marketing channels, and therefore are not subjected to reliable methods of veterinary inspection. While this scenario might be more typical of developing countries, the situation exists to some extent in most countries of the world.

The knowledge of modes of transmission of Trichinella to domestic pigs, allows pig farmers/producers to design management systems which prevent or drastically reduce the risk of exposure. By following a series of good management practices, combined with documentation of these practices and regular official control to verify that these practices are effective, it is possible to certify the safety of pork without subsequent slaughter inspection or further processing. There are minimal requirements that need to be met for livestock to be considered Trichinella-free.

Programmes, which allow certification of pigs as free from Trichinella larvae should be administratively organized to allow proper documentation of certified herds. This is by developing a system of documentation of Trichinella-free production practices, which addresses issue certifications and maintain records of certified farms; periodically conducting spot audits of certified producers to assure the integrity of the system; and conducting periodic serology testing of pigs originating from certified farms to verify absence of infection (Forbes and Gajadhar 1999; Gajadhar and Forbes 2002; Forbes et al. 2005).

Recent legislation of the EU (European Commission 2005) describes requirements for certifying pigs from farms or categories of farms which raise pigs under certain conditions such as confinement housing, as free from risk of Trichinella infection. Pigs from these farms are exempt from requirements for Trichinella inspection at slaughter. In the USA, a system for certifying pig farms free from risk of exposure to Trichinella is in a pilot phase. This programme emphasizes auditing of good production practices which document the absence of risk factors that would exposure pigs to Trichinella in feed, rodents and wildlife (www.aphis.usda.gov/vs/trichinae). As part of the audit requirements, farms must maintain accurate records of animal movement, animal disposal, and rodent control logs. Further, it provides for education of veterinary practitioners who are responsible for conducting regular audits of farms seeking and maintaining Trichinella-free certification.

The International Commission on Trichinellosis does not endorse any programme for assuring pigs to be free from Trichinella based on geographic boundaries such as a region, state or country (OIE 2006). Indeed, Trichinella occurs in a wide range of wildlife reservoirs both in terrestrial and marine mammals and birds. The absence of Trichinella infection cannot be reliably documented in these various species.