Biology of Trichinella spiralis

Trichinella spiralis is known to have a broad vertebrate host range and can infect all vertebrates tested, except Chinese hamsters. Trichinella spiralis has also been documented in all continents of the world (reviewed by Despommier 1983). The lifecycle of T. spiralis is characteristic of nematodes and includes an adult stage followed by four larval stages (Figure 21.2). Trichinella spiralis spends the majority of its life at larval stage 1 (L1) which is also the infective stage. When any warm-blooded vertebrate consumes an infected muscle tissue the consumer can get trichinellosis.

Following ingestion of infected muscle tissue, the acidic environment together with pepsin in the stomach, releases the L1 larvae within minutes. No visible changes occur in the larvae during this phase. The free larvae migrate rapidly to the small intestine and enter the columnar epithelial cells; this process may take as little as 10 min (Despommier et al. 1978; Wright 1979). At this stage the larvae are approximately 1mm long and have a diameter of approximately 0.040mm. Since each columnar cell measures 0.032 x 0.0085mm, the nematode occupies about 117 columnar cells. After penetration, a fusion of the columnar cell membranes occurs to form a syncytium (Wright 1979). The mechanism(s) for the penetration and generation of the syncytium is (are) not known.

The L1 larvae then rapidly undergo three molts to become L4 larvae after 30h of infection (Wright 1979). Moulting in T. spiralis seems to be similar to that seen in other worms, at least from the L1 to L2 stages where the skin is physically removed and the worms literally crawl out of it. Similarly, T. spiralis probably needs a physical barrier to rub against in order to remove parts of its outer cuticle. Sexually mature adults are observed at 1h, to up to several months, after the L4 stage is reached (with a peak at five days). In the adult stage, a difference in size between the females and males is seen. The females, which now occupy 415-425 columnar cells, are approximately 3mm long, while the males are still approximately 1mm long (Wright 1979; Stewart and Giannini 1982). Other important changes which occur are the development of the male genitalia and the female cloacal opening which are not present in the L1 stage. Changes can be seen in the L2 stage and the organs are developed in the adult stage (Burnham and Despommier 1984). The two outer layers of the cuticle also disappear in the L2 stage

Figure 21.2 The lifecycle of Trichinella spiralis. Digestion of infected muscle tissue (top) results in release of the first larval stage. The larva then migrates and penetrates the columnar epithelial cells (left). They mate as adults after four molts, 5-6 days post-infection (PI) (bottom). The newborn larvae migrate to the lymphatic system and are passively transported to the skeletal muscle cells which they mechanically penetrate (right). Approximately 20 days after the penetration (PP) of the skeletal muscle cell, a nurse cell is formed, which can support the parasite for several years or until the tissue gets digested again (top, reviewed by Despommier 1983).

Figure 21.2 The lifecycle of Trichinella spiralis. Digestion of infected muscle tissue (top) results in release of the first larval stage. The larva then migrates and penetrates the columnar epithelial cells (left). They mate as adults after four molts, 5-6 days post-infection (PI) (bottom). The newborn larvae migrate to the lymphatic system and are passively transported to the skeletal muscle cells which they mechanically penetrate (right). Approximately 20 days after the penetration (PP) of the skeletal muscle cell, a nurse cell is formed, which can support the parasite for several years or until the tissue gets digested again (top, reviewed by Despommier 1983).

(Stewart et al. 1987), which mediates a change in the surface coat. Different patterns of surface molecules are seen in all stages of the nematode (Philipp et al. 1980; Parkhouse and Ortega-Pierres 1984). Other changes occurring include development of hypodermal gland cells. These cells are organized in four rows, two dorsal and two ventral, which run almost for the whole length of the nematode. The function of these subcuticular cells is unknown (Wright 1979).

Mating occurs 37-40 h after infection but has a peak at day 5. It is believed that a pheromone system is in operation when the nematodes are finding each other (Belosevic and Dick 1980). Embryogenesis takes approximately 90h and the release of the newborn larvae can occur as early as five days, but with a peak between days 8 and 9 after infection. Fecundity varies depending on the infectivity of the Trichinella isolate, the number of infecting nematodes, and the immunological status of the infected animal. Values in mice vary between 14 and 600 newborn larvae per L1 larva (reviewed by Despommier 1983).

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The newborn larvae migrate from the lumen of the columnar epithelial cells to the lamina propria of the villus tissue. They then can penetrate the lymphatic system and travel via the thoracic duct to the blood system (Harley and Gallicchico 1971; Despommier et al. 1978) where they are passively transported to their target cells, the striated skeletal muscle cells (Dennis et al. 1970). It is not known how the nematode recognizes these cells, or how penetration occurs but it is thought that the nematode mechanically penetrates the muscle cells, then migrates several worm lengths away from its point of entry (Despommier 1993). The worm now starts a visible period of growth and development without moulting to become a mature infective L1 larva. The volume increases by more than 270 times from day 5 to day 20, when its differentiation to L1 is complete. The main organ that is developed during this stage is the stichosome, an organ that has been proposed to be used for secretion. Each stichosome consists of cells, stichocytes, which contain secretory granules. The number of stichocytes in the stichosome increases from about 20 on day 1 after entering the cell to a final 50-55 at day 20. The stichocytes differentiate into either cells containing 10-15 alfa-granules, found in the posterior region, or cells containing 35-40 beta-granules, found within the anterior region (Despommier and Muller 1976; Takahashi et al. 1989). Mature stichocytes are about 0.025mm in diameter and posses, a single nucleus, mitochondrion, Golgi complex, rough endoplasmic reticulum, and either approximately 2000 alfa-granules or approximately 3000 beta-granules (Despommier and Muller 1976). The granules are surrounded by a single membrane and contain molecules which are recognized by antibodies raised against surface antigens from the nematode, suggesting a secretory role for the stichocytes (Despommier and Muller 1976; Grencis et al. 1986; Takahashi et al. 1989).

The muscles most frequently involved in trichinellosis are the fibres nearest to the site of attachment of the tongue, larynx, diaphragm, and neck. Once the parasite has invaded the muscle cell, the vascular network surrounding the muscle increases, a phenomenon which is thought to be induced by the parasite (Baruch and Despommier 1991). After 20 days of nematode development in the muscle cell, the parasite has developed a stable environment, a nurse cell (Despommier et al. 1975). The nurse cell continues to grow for another 30 days and the thickness of the capsule increases until day 50 after penetration of the nematode into the muscle cell (Teppema et al. 1973). Between days 10 and 14 after penetration of the muscle cell, the final two layers of the nematode cuticle, 3 and 4, can be observed for the first time. It is in the first 20 days during exponential growth of the larvae that most changes occur, both in macromolecules and in the microanatomical structure observed in the infected muscle cell (Jasmer 1990). There is a disappearance of myofilaments in the infected muscle cells. This is correlated with a decrease in the content of muscle-specific proteins, such as myosin heavy chain, a-actin, and tropomysin (Teppema et al. 1973; Jasmer 1990). There is also an increase in the number of organelles, including smooth and rough endoplasmic reticulum, free ribosomes, mitochondria which appear vacuolated, Golgi apparatus, and lysosomes (Maier and Zaiman 1966; Despommier 1975). The DNA content in the infected muscle cell nuclei increases approximately two fold compared with the non-infected muscle cell (Jasmer 1993). These data indicate that the infected muscle cell has left the normal differentiated state and re-entered the cell cycle in order to undergo S-phase in which the DNA content multiplies for the later division into two cells. Since no division of the infected muscle cell has been observed, it is thought that the cell stops at the G2/M-phase (Jasmer 1993). The levels of specific RNA

transcripts encoding several muscle-specific proteins are decreased to <0.1% when compared with non-infected muscle cells (Jasmer et al. 1991). Transcript levels of the myogenic regulatory proteins MyoD and myogenin are reduced to <1% and <10%, respectively, while the level of the negative regulator Id is increased to approximately 250% (Jasmer 1993).

The decreased expression of the muscle-specific proteins has been suggested to be due to the down regulation of the myogenic regulatory proteins, MyoD and myogenin, in the infected muscle cell (Jasmer 1993; Lindh 1996). The changes in the expression patterns of the myogenic regulatory proteins could be due to a secreted molecule from the nematode, which might interact with the host molecules and be responsible for their different expression pattern (Ko et al. 1992, 1994a,b; Jasmer 1993; Lindh 1996). The increase in the number of different organelles together with the increase in DNA synthesis to approximately 4N has led to the following suggestion: the differentiated muscle cell has left the differentiated state because of secreted products from the nematode and entered the cell cycle again (Stewart 1983; Jasmer 1993). Evidence that the dedifferentiation process seen in the infected muscle cells is due to ES products comes from experiments in which ES was collected from L1-stage larvae and then reintroduced into healthy muscle cells (Ko et al. 1992). The reorganization seen in these muscle cells was similar to those seen in muscle cells infected with infective larvae (Ko et al. 1992, 1994a,b; Jasmer et al. 1994). A possible negative regulator to muscle differentiation has also been identified and found to be secreted from the L1 larva into the muscle cell (Lindh 1996).

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