47 Toxoplasmosis, sarcocystosis, isosporosis, and cyclosporosis

Toxoplasmosis is a protozoan disease caused by Toxoplasma gondii. It is widely prevalent in humans and animals throughout the world, especially in the western hemisphere. Virtually all warm-blooded animals can act as intermediate hosts but the life cycle is completed only in cats, the definitive host. Cats excrete the resistant stage of T. gondii (oocysts) in faeces, and oocysts can survive in the environment for months. Humans become infected congenitally, by ingesting undercooked infected meat, or by ingesting food and water contaminated with oocysts from cat faeces. It can cause mental retardation and loss of vision in congenitally infected children and deaths in immunosuppressed patients, especially those with Acquired immunodeficiency syndrome (AIDS). There is no vaccine to control toxoplasmosis in humans at the present time but one is available for reduction of fetal losses in sheep.

Toxoplasma gondii was discovered in 1908 in Tunisia in a rodent, Ctenodoctylus gundi, and in a laboratory rabbit in São Paulo, Brazil (Table 47.1). The name Toxoplasma (toxon = arc, plasma = form) is derived from the crescent shape of the tachyzoite stage, and the host, gundi. The medical importance of T. gondii was not discovered until late 1930s (Wolf et al. 1939). The development of a serological test for toxoplasmosis in 1948 led to the findings that it was a common infection of humans throughout the world (Table 47.1).

While considerable progress on the characterization of the disease in humans and animals was made between 1940 and 1960, the main routes of transmission remained a mystery. Congenital transmission occurred too rarely to explain widespread infection in humans and animals. In the 1960s it was found that organisms from tissue cysts could survive digestive enzymes and that humans can become infected by ingesting undercooked infected meat.

While congenital transmission and carnivorism partially explain transmission of T. gondii, these routes cannot explain the widespread T. gondii infection in vegetarians and in herbivores. Prevalence rates for T. gondii in strict vegetarians were found to be similar to those in non-vegetarians. Fresh excretions and secretions of animals which had even overwhelming infections proved essentially negative for T. gondii when tested in mice. Attempts to transmit T. gondii via arthropods were essentially unsuccessful.

The mystery of transmission was resolved when a resistant form of T. gondii was discovered in feline faeces and the coccidian phase of its life cycle was discovered in 1970 (Table 47.1).

Toxoplasma gondii (Nicolle and Manceaux 1908) Nicolle and Manceaux 1909 is a coccidian parasite of cats with warm-blooded animals as intermediate hosts. Coccidia are among the most important parasites of animals. Traditionally, all coccidia of veterinary importance were classified under the family Eimeriidae, Michin, 1903. Classification was based on the structure of the oocyst. Oocysts with four sporocysts, each with two sporozoites (total eight sporozoites) are classified as Eimeria. Oocysts containing two sporocysts, each with four sporozoites, were classified historically as Isospora. After the discovery of the life cycle of T. gondii, several other genera (Sarcocystis, Besnoitia, Hammondia, Neospora, Frenkelia) were found to have isosporan oocysts with two sporocysts and eight sporozoites.

Toxoplasma gondii and related genera discussed in the chapter are classified as follows:

Opinions differ regarding the further classification of T. gondii into families and subfamilies. It has been classified in the family Eimeriidae (Minchin 1903), Sarcocystidae (Poche 1913), or Toxoplasmatidae (Biocca 1956) by various authorities.

There are three infectious stages of T. gondii (Fig. 47.1): the tachyzoites (in groups), the bradyzoites (in tissue cysts), and the sporozoites (in oocysts) (Frenkel 1973).

The tachyzoite is often crescent-shaped and is approximately 2 × 6 μm (Fig. 47.2). Its anterior (conoidal) end is pointed and its posterior end is round. It has a pellicle (outer covering), polar ring, conoid, rhoptries, micronemes, apicoplast, mitochondria, subpellicular microtubules, endoplasmic reticulum, Golgi apparatus, ribosomes, rough surface endoplasmic reticulum, micropore, and a well defined nucleus (Fig. 47.3). The nucleus is usually situated toward the posterior end or in the central area of the cell (Dubey 1977, 1993; Ferguson and Dubremetz 2007).

The pellicle consists of three membranes. The inner membrane complex is discontinuous at three points: the anterior end (polar ring), the lateral edge (micropore), and toward the posterior end. The polar ring is an osmiophilic thickening of the inner membrane at the anterior end of the tachyzoite. The polar ring encircles a cylindrical, truncated cone (the conoid) which consists of 6–8 fibrillar elements wound like a compressed spring. Twenty-two subpellicular microtubules originate from the anterior end and run longitudinally almost the entire length of the cell. Terminating within the conoid are 4–10 club-shaped organelles called rhoptries (Fig. 47.3). The rhoptries are gland-like structures, often labrinthine, with an anterior narrow neck up to 2.5 μm long. Their sac-like posterior end terminates anterior to the nucleus. Micronemes are rod shaped electron dense structures which occur at the anterior end of the parasite (Dubremetz and Ferguson 2007  ).

The functions of the conoid, rhoptries, and micronemes are not fully known but are involved in penetration and successful location within a host cell (Boothryod and Dubremetz 2008). The conoid is probably associated with the penetration of the tachyzoite through the membrane of the host cell. It can rotate, tilt, extend, and retract as the parasite searches for a host cell. Toxoplasma gondii can move by gliding, undulating, and rotating. Rhoptries have a secretory function associated with host cell penetration, secreting their contents through the conoid to the exterior. The microtubules probably provide the cytoskeleton.

The tachyzoite enters the host cell by active penetration of the host cell membrane. After entering the host cell the tachyzoite becomes ovoid in shape and becomes surrounded by a parasitophorous vacuole (PV). It has been suggested that the PV is derived from both the parasite and the host. Numerous intravacuolar tubules connect the parasitophorous vacuolar membrane to the parasite pellicle.

The tachyzoite multiplies asexually within the host cell by repeated endodyogeny. Endodyogeny (endo = inside, dyo = two, geny = progeny) is a specialized form of reproduction in which two progeny form within the parent parasite, consuming it in the process. Tachyzoites continue to divide by endodyogeny until the host cell is filled with parasites (Fig. 47.3B).

After a few divisions, T. gondii encysts to form tissue cysts (Fig. 47.4). Tissue cysts grow and remain intracellular as the bradyzoites (encysted T. gondii) divide by endodyogeny. Tissue cysts vary in size. Young tissue cysts may be as small as 5 μm and contain only two bradyzoites, while older ones may contain hundreds of organisms (Dubey et al. 1998) (Fig. 47.4). Tissue cysts in brain are often circular and rarely reach a diameter of 70 μm whereas intramuscular cysts are elongated and may be 100 μm long. Although tissue cysts may develop in visceral organs, including lungs, liver, and kidneys, they are more prevalent in the neural and muscular tissues, including the brain, eye, skeletal, and cardiac muscle. Intact tissue cysts probably do not cause any harm and can persist for the life of the host.

The tissue cyst wall is elastic, thin (< 0.5 μm) and argyrophilic (Fig. 47.4B). The bradyzoites are approximately 7 × 1.5 μm. Bradyzoites differ structurally only slightly from tachyzoites. They have a nucleus situated toward the posterior end, whereas the nucleus in tachyzoites is more centrally located. The contents of rhoptries in bradyzoites in older tissue cysts are electron dense (Fig. 46.5). Bradyzoites contain several amylopectin granules which stain red with periodic acid–Schiff (PAS) reagent (Fig. 47.4C); such material is either in discrete particles or absent in tachyzoites. Bradyzoites are more slender than are tachyzoites. Bradyzoites are less susceptible to destruction by proteolytic enzymes than are tachyzoites.

Cats excrete oocysts after ingesting tachyzoites, bradyzoites, or sporozoites. Prepatent periods (time to the shedding of oocysts after initial infection) and frequency of oocyst shedding vary according to the stage of T. gondii ingested. Prepatent periods are 3–10 days after ingesting tissue cysts and 18 days or more after ingesting tachyzoites or oocysts (Dubey 2005b). Intermediate prepatent periods of 11–17 days maybe associated with ingestion of transitional stages between tachyzoites and bradyzoites (Dubey 2005b). Fewer than 50% of cats shed oocysts after ingesting tachyzoites or oocysts, whereas nearly all cats shed oocysts after ingesting tissue cysts. From an epidemiological view point, cats are likely to shed oocysts after ingesting tissues of infected rodents, irrespective of their clinical status.

After the ingestion of tissue cysts by cats, the cyst wall is dissolved by the proteolytic enzymes in the stomach and small intestine. The released bradyzoites penetrate the epithelial cells of the small intestine and initiate development of numerous generations of T. gondii (Fig. 47.5A, B). Five morphologically distinct types (A to E) of T. gondii develop in intestinal epithelial cells before gametogony begins. Types A to E divide asexually by endodyogeny, endopolygeny, or schizogony (division into more than two organisms) (Dubey and Frenkel 1972; Speer and Dubey 2005).

Mature male gamonts are ovoid to ellipsoidal in shape. Each microgamete has two flagella (Fig. 47.6C). The microgametes swim to and penetrate a mature macrogamete. After penetration, oocyst wall formation begins around the fertilized gamete. When they are mature, oocysts are discharged into the intestinal lumen by the rupture of intestinal epithelial cells.

Unsporulated oocysts are subspherical to spherical and are 10 × 12 μm in diameter (Fig. 47.8A). The oocyst wall contains two colourless layers. The sporont almost fills the oocyst, and sporulation occurs outside the cat within 1–5 days, depending upon aeration and temperature.

Sporulated oocysts are subspherical to ellipsoidal and are 11 × 13 μm in diameter. Each sporulated ooccyst contains two ellipsoidal sporocysts without a Stieda body. Sporocysts measure 6 × 8 μm (Fig. 47.8B). There are four sutures with lip-like thickenings in the sporocyst wall (Fig. 47.8B); these sutures open during excystation of the sporozoites. A sporocyst residuum is present. There is no oocyst residuum. Each sporocyst contains four sporozoites. The sporozoites are 2 × 6–8 μm in size with a subterminal to central nucleus and a few PAS-positive granules in the cytoplasm (Fig. 47.8B).

As the enteroepithelial cycle progresses, bradyzoites penetrate the lamina propria of the feline intestine and multiply as tachyzoites. Within a few hours after infection of cats, T. gondii may disseminate to extraintestinal tissues. Toxoplasma gondii persists in intestinal and extraintestinal tissues of cats for at least several months, if not for the life of the cat.

Toxoplasma gondii is biologically adapted to transmission by carnivorism in cats (Frenkel et al. 1970). By the oral route, bradyzoites are more infective to cats than mice and oocysts are more infective to mice and pigs than cats (Dubey 2001, 2006).

Toxoplasma gondii has not been grown in cell-free media. Toxoplasma gondii can be cultivated in laboratory animals, chick embryos, and cell cultures. Mice, hamsters, guinea-pigs, and rabbits are all susceptible but mice are generally used as hosts because they are more susceptible than the others and are not naturally infected when raised in the laboratory on commercial dry food free of cat faeces.

Tachyzoites of some strains of T. gondii grow in the peritoneal cavity of mice, sometimes producing ascites, and also grow in most other tissues after intraperitoneal inoculation with any of the three infectious stages of T. gondii. Virulent strains usually produce illness in mice and sometimes kill them within 1–2 weeks. Most strains of T. gondii do not kill mice.

Toxoplasma gondii tachyzoites will multiply in many cell lines in cell cultures (Fig. 47.9). Although tissue cysts can develop in cell cultures with most strains of T. gondii, the yield is lower than that produced by infection in mice.

Tissue cysts are obtained by injecting tachyzoites, bradyzoites, or oocysts into mice. To obtain tissue cysts from mice inoculated with a virulent strain, it is necessary to administer anti-T. gondii chemotherapy to prevent death from acute toxoplasmosis before tissue cysts form. Sulphadiazine is effective in controlling the acute stages of toxoplasmosis in mice. Tissue cysts are prominent in the mouse brain about 8 weeks after infection (Fig. 47.10).

Enteroepithelial stages of T. gondii have not yet been cultivated in vitro.

Oocysts can be obtained by feeding tissue cysts from infected mice to T. gondii-free cats.

Toxoplasma gondii nucleus is haploid except during the sexual division in the intestine of the cat (Pfefferkorn 1990). Sporozoites are the results of meiosis and seem to follow classical Mendelian laws. The total haploid genome contains 14 chromosomes, 7,793 genes, with total genome size of 63,495,144 base pairs (Khan et al. 2007). It is an unusual parasite because of its broad host range and with only one species in the genus. Prior to the development of genetic markers, T. gondii isolates were grouped by their virulence to outbred mice (Dardé et al. 2007). Based on restriction fragment length polymorphism (RFLP), Howe and Sibley (1995  ) classified T. gondii into 3 genetic Types (I, II, III) and linked mouse virulence to genetic type. They proposed that Type I isolates were 100% lethal to mice, irrespective of the dose, and that Types II and III generally were avirulent for mice (Howe et al. 1996). Furthermore, until recently, T gondii was considered to be clonal with low genetic variability, and strains isolated from asymptomatic hosts were considered avirulent. However, recent studies have indicated that most T. gondii isolates from asymptomatic chickens from Brazil and Colombia in South America were virulent for mice, were non-clonal, Type II was absent, and there was marked genetic variability (Dubey et al. 2002; Lehmann et al. 2006). In humans in French Guiana and Suriname, severe cases of toxoplasmosis in immunocompetent patients have been related to mouse-virulent T. gondii strains with atypical genotypes (Carme et al. 2002; Demar et al. 2007). Circumstantial evidence suggests that certain genetic types of T. gondii may be associated with clinical ocular toxoplasmosis in humans (Khan et al. 2006). It has been suggested that Type I isolates or recombinants of Types I and III are more likely to result in clinical toxoplasmosis, but genetic characterization has been limited essentially to isolates from patients ill with toxoplasmosis. There is very little information regarding the genetic diversity of T. gondii isolates circulating in the general human population. Therefore, we must be cautious in claiming a linkage between parasite genotypes and disease presentations without clear and

discerning information regarding the parasite’s biology in the human population and environment. In my opinion, all isolates of T. gondii must be considered potentially pathogenic and laboratory workers and public health persons must take appropriate precautions while handling materials potentially infected with T. gondii (Dubey 2009).

Toxoplasma gondii usually parasitizes the host (both definitive and intermediate) without producing clinical signs. Only rarely does it cause severe clinical manifestations. The majority of natural infections are probably acquired by ingestion of tissue cysts in infected meat or oocysts in food or water contaminated with cat faeces. The bradyzoites from the tissue cysts or sporozoites from the oocyst penetrate the intestinal epithelial cells and multiply (Fig. 47.11A). Toxoplasma gondii may spread first to the mesenteric lymph nodes (Fig. 47.11B) and then to distant organs by invasion of lymph and blood. An infected host may die because of necrosis of intestine and mesenteric lymph nodes before other organs are severely damaged. Focal areas of necrosis may develop in many organs. The clinical picture is determined by the extent of injury to organs, especially vital organs such as the eye, heart, and adrenal glands. Necrosis is caused by the intracellular growth of tachyzoites. Toxoplasma gondii does not produce a toxin.

In those hosts which develop disease, the host may die of acute toxoplasmosis but much more often recovers with the acquisition of immunity (Frenkel 1973). In the recovering individual inflammation usually develops in sites where initially there was necrosis. By about the third week after infection, T. gondii tachyzoites begin to disappear from the visceral tissues and may localize in tissue cysts in neural and muscular tissues. Toxoplasma gondii tachyzoites may persist longer in the spinal cord and brain than in visceral tissues because immunity there is less effective than in neural organs. Toxoplasma gondii tachyzoites can persist in the placenta for months after the initial infection of the dam. How T. gondii is destroyed in immune cells is not completely known. All extracellular forms of the parasite are directly affected by antibody but intracellular forms are not. It is believed that cellular factors including lymphocytes and lymphokines are more important than humoral ones in immune-mediated destruction of T. gondii (Frenkel 1973; Gazzinelli et al. 1993). Under experimental conditions, infection with avirulent strains protects the host from damage but does not prevent infection with more virulent strains. In most instances, immunity following a natural T. gondii infection persists for the life of the host.

Immunity does not eliminate infection. Toxoplasma gondii tissue cysts persist several years after acute infection. The fate of tissue cysts is not fully known. Whether bradyzoites can form new cysts directly without transforming into tachyzoites is not known but likely (Dubey 2005b). It has been proposed that tissue cysts may at times rupture during the life of the host. The released bradyzoites may be destroyed by the host’s immune responses. The reaction may cause local necrosis accompanied by inflammation. Hypersensitivity plays a major role in such reactions. After such events, inflammation usually again subsides with no local renewed multiplication of T. gondii in the tissue; however, occasionally there may be formation of new tissue cysts.

In immunosuppressed patients, such as those given large doses of immunosuppressive agents in preparation for organ transplants and in those with AIDS, rupture of a tissue cyst may result in transformation of bradyzoites into tachyzoites and renewed multiplication (Fig. 47.12). The immunosuppressed host may die from toxoplasmosis unless treated. It is not known how corticosteroids cause relapse but it is unlikely that they directly cause rupture of the tissue cysts.

Pathogenicity of T. gondii is determined by the virulence of the strain and the susceptibility of the host species. Toxoplasma gondii strains may vary in their pathogenicity in a given host. Certain strains of mice are more susceptible than others and the severity of infection in individual mice within the same strain may vary. Certain species are genetically resistant to clinical toxoplasmosis. For example, adult rats do not become ill while the young rats can die because of toxoplasmosis. Mice of any age are susceptible to clinical T. gondii infection. Adult dogs, like adult rats, are resistant, whereas puppies are fully susceptible. Cattle and horses are among the hosts more resistant to clinical toxoplasmosis whereas certain marsupials and New World monkeys are the most susceptible. Nothing is known concerning genetic-related susceptibility to clinical toxoplasmosis in higher mammals, including humans.

Various factors vaguely classified as stress may affect T. gondii infection in a host. More severe infections are found in pregnant or lactating mice than in non-lactating mice. Concomitant infection may make the host more susceptible or resistant to T. gondii infection.

Toxoplasma gondii infection is widespread among humans and its prevalence varies widely from place to place (Dubey and Beattie 1988; Dubey 2009). In the USA and the UK it is estimated that about 16–40% of people are infected, whereas in Central and South America and continental Europe estimates of infection range from 50–80%.

Most infections in humans are asymptomatic but at times the parasite can produce devastating disease. Infection may be congenitally or postnatally acquired.

Congenital infection occurs generally when a woman becomes infected during pregnancy and the severity of disease may depend upon the stage of pregnancy when the woman becomes infected (Elbez-Rubinstein et al. 2009) (Table 47.2). While the mother rarely has symptoms of infection, she does have a temporary parasitaemia. Focal lesions develop in the placenta and the fetus may become infected. At first there is generalized infection in the fetus. Later, infection is cleared from the visceral tissues and may localize in the central nervous system. A wide spectrum of clinical disease occurs in

congenitally infected children. Mild disease may consist of slightly diminished vision only, whereas severely diseased children may have the full tetrad of signs: retinochoroiditis and hydrocephalus (Fig. 47.13), convulsions and intracerebral calcification (Fig. 47.14). Of these, hydrocephalus is the least common but most dramatic lesion of toxoplasmosis. This lesion is unique to congenitally acquired toxoplasmosis in humans and has not been reported in other animals.

By far the mo`st common sequel of congenital toxoplasmosis is ocular disease (Guerina et al. 1994; McAuley et al. 1994; Remington et al. 2006). Except for the occasional involvement of an entire eye, in virtually all cases the disease is confined to the posterior chamber. Toxoplasma gondii proliferates in the retina and this leads to inflammation in the choroid. Therefore, the disease is correctly designated as retinochoroiditis. In humans the characteristic lesions of ocular toxoplasmosis in the acute or subacute stage of inflammation appear as yellowish-white, cotton-like patches in the fundus. The lesions may be single or multiple and may involve one or both eyes (Dutton 1989). During the acute stage, inflammatory exudate may cloud the vitreous fluid and may be so dense as to preclude visualization of the fundus by ophthalmoscope examination. As the inflammation subsides, the vitreous clears and the diseased retina and choroid can be seen through the ophthalmoscope. Retinal lesions may be single or multifocal, small, grey areas of active retinitis with minimal oedema and reaction in the vitreous humour. The punctuate lesions are usually harmless unless they are located in a macular area (Fig. 47.15). Although severe infections may be detected at birth, milder infections may go undetected until they flare up in adulthood.

The socio-economic impact of toxoplasmosis in human suffering and the cost of care of sick children, especially those with mental retardation and blindness, are enormous (Roberts and Frenkel 1990; Roberts et al. 1994). The testing of all pregnant women for T. gondii infection is compulsory and all pregnant women are tested serologically on their first visit to their gynaecologist. Women with T. gondii antibodies are not tested further. Seronegative women are tested monthly and they are treated for toxoplasmosis if they acquire T. gondii antibodies during pregnancy. Studies from France and Austria indicate that treatment of women during pregnancy reduces fetal damage. The cost benefits of such mass screening are being debated in many countries (Dubey and Beattie 1988; Lebech and Petersen 1992; Remington et al. 2006; Petersen 2007).

Most people infected after birth are asymptomatic (Montoya and Liesenfeld 2004; Remington et al. 2006). However, some develop a mild disease or in rare cases, a more severe systemic illness and even fatal disease, including pulmonary and multivisceral involvement, possibly from more virulent types of the organism (Carme et al. 2002; Demar et al. 2007). Once infected, people are believed to remain infected for life. Unless immunosuppression occurs and the organism reactivates, people usually remain asymptomatic. However, research is ongoing on whether chronic T. gondii infection has an effect on reaction time, tendency for accidents, behavior, and mental illness (Lafferty 2006; Dubey and Jones 2008).

Postnatally acquired infection may be localized or generalized (Table 47.3). Oocyst-transmitted infections may be more severe than tissue cyst-induced infections. Lymphadenitis is the most frequently observed clinical form of toxoplasmosis in humans. Although any node may be involved, the most frequently involved are the deep cervical nodes. These nodes when infected are tender, discrete but not painful, and the infections resolve spontaneously in weeks or months. Lymphadenopathy may be associated with fever, malaise, fatigue, muscle pain, sore throat, and headache. Although the condition may be benign, its diagnosis is vital in pregnant women because of the risk to the fetus.

Until recently, most of toxoplasmic retinochoroiditis was thought to be congenital (Holland 2003  ). Ophalmologists from Brazil first reported retinochoroiditis in multiple siblings (Silveria et al. 1988). These findings have now been amply confirmed (Burnett et al. 1998  ). In a largest outbreak of human toxoplasmosis epidemiologically linked to drinking water from a municipal water reservoir in Vancouver, British Columbia, Canada, 20 of 100 patients had acquired retinochoroiditis (Bowie et al. 1997; Burnett et al. 1998). Lymphardenitis was found in 51 of these 100 patients (Bowie et al. 1997).

Toxoplasmosis ranks high in the list of diseases which lead to death of patients with AIDS; approximately 10% of AIDS patients in the USA and up to 30% in Europe are estimated to die from toxoplasmosis (Luft and Remington 1992; Luft et al. 1993; Rabaud et al. 1994). Clinically, patients may have headache, disorientation, drowsiness, hemiparesis, reflex changes, and convulsions, and many become comatose. Diagnosis is aided by serological examination. However, in immunosuppressed patients both inflammatory signs and antibody production may be suppressed, thus making the diagnosis very difficult. Although in AIDS patients any organ may be involved, including the testis, dermis, and the spinal cord, infection of the brain is most frequent. In the brain, the predominant lesion is necrosis, especially of the thalamus. In most AIDS patients, the disease is reactivation of latent T. gondii infection because of immunosuppressive effects of the human immunodeficiency virus infection.

By using CT scan, lesions were localized in decreasing order of frequency in cortico-medullary, white matter, basal ganglions, cortex and posterior fossa (Renold et al. 1992). Macroscopically, unilateral or bilateral areas of discolouration indicative of necrosis and haemorrhage were noticed. Microscopically, encephalitis involving many areas is the predominant lesion and the difference may vary depending on whether the patient has received anti-toxoplasma therapy. In untreated patients, the lesion involves a central area of necrosis with degenerating organisms, surrounded by an inflammatory zone with oedema, perivascular infiltration of inflammatory cells and haemorrhage. Toxoplasma gondii are more numerous in this peripheral zone surrounding healthy and inflamed tissue. The lesion may be small or the size of a tennis ball and the contents may vary from fluid to solid. Microabscesses are more common in treated patients. In active lesions, numerous tachyzoites are found destroying host tissue. In subacute cases and treated patients, glial nodules predominate. Patients should be treated empirically for toxoplasmic encephalitis based on clinical and neurological findings and presence of T. gondii antibodies because a specific diagnosis may not be possible without a biopsy, which is now rarely indicated. Since the years that prophylaxis and highly active antiretroviral therapy became widely used (mid 1990s in most developed countries), the incidence and deaths associated with toxoplasmic encephalitis have declined markedly.

Transplantation of infected organs or transfusion of infected leukocytes can initiate fatal infection in a seronegative recipient receiving immunosuppressive therapy. Transplantation of non-infected organs and leukocyte transfusion can also activate latent infection in a seropositive recipient receiving immunotherapy. Ordinary blood transfusion is virtually free from danger, but transfusion of packed leukocytes and transplantation of bone marrow have caused toxoplasmosis. It seems that the danger of transplanting an organ from a seropositive donor into a seronegative recipient is greater than that of transplanting an organ from a seronegative donor into a seropositive recipient. Recipients of heart and heart lung are more likely to have symptomatic infection than kidney or liver transplant patients (Wreghitt and Joynson 2001).

Malignancies or immunosuppressive treatment of malignancies can reactivate latent toxolasmosis. Toxoplasmosis has been reported most commonly in patients treated for Hodgkin’s disease. Untreated Hodgkin’s disease is rarely associated with clinical toxoplasmosis. A variety of malignancies including lymphoma, leukaemia, myelomna can reactive toxoplasmosis but there are rare reports of toxoplasmosis associated with solid tumors (Wreghitt and Joynson 2001).

Toxoplasma gondii is capable of causing severe disease in animals other than humans. Among livestock, great losses occur in sheep and goats. Toxoplasma gondii causes early embryonic death and resorption, fetal death and mummification, abortion, stillbirth, and neonatal death. Toxoplasmosis-induced abortion can occur in ewes of all ages. Infected lambs that survive the first week after birth grow normally. Abortion occurs in ewes that acquire infection during pregnancy. Therefore, ewes which have aborted should be saved for future breeding. Fatal toxoplasmosis has been reported in pigs, dogs, cats, rabbits, birds, and many species of wildlife (Dubey and Beattie 1988; Dubey 2009).

Cattle and horses are more resistant to clinical toxoplasmosis than any other species of livestock. Although both cattle and horses have been found infected with T. gondii, there is no documented report of clinical toxoplasmosis in horses or cattle. Toxoplasmosis is most severe in certain species of Australian marsupials and New World monkeys (Dubey and Beattie 1988). It can cause severe blindness in canaries and finches, and wallabies (Dubey 2002; Dubey and Crutchley 2008).

Recent findings of high prevalence of T. gondii in free-living marine mammals and mortality associated with protozoal encephalitis in sea otters in the USA has raised concerns for environmental contamination of the environment with T. gondii oocysts (Kreuder et al. 2003; Thomas et al. 2007; Dubey and Jones 2008).

Diagnosis is made by biological, serological, histological, or molecular methods or by some combination of them. Clinical symptoms of toxoplasmosis are non-specific and toxoplasmosis in fact mimics several other infectious diseases.

Toxoplasma gondii can be isolated from patients by inoculation of laboratory animals and tissue cultures with secretions, excretions, body fluids, and tissues taken by biopsy ante-mortem or tissues with macroscopic lesions taken post-mortem.

Detection of T. gondii antibody in patients may aid diagnosis. There are numerous serological procedures used to detect humoral antibodies; these include the Sabin–Feldman dye test, the indirect haemaglutination assay, the indirect fluorescent antibody assay (IFA), the direct agglutination test (DAT), the latex agglutination test, the enzyme-linked immunoabsorbent assay (ELISA), and the immunoabsorbent agglutination assay test (IAAT). The IFA, IAAT, and ELISA have been modified to detect IgM antibodies. The IgM antibodies appear sooner than the IgG antibodies but IgM antibodies also disappear faster than IgG antibodies.

The result of examining one positive serum sample only establishes that the host has been infected at some time in the past. It is best to collect two samples on the same individual. A 16-fold higher antibody titre in a serum taken 2–4 weeks after the first serum was collected indicates an acute acquired infection. A high antibody titre sometimes persists for months and a rise may not be associated with clinical symptoms. As indicated earlier, most acquired infections in humans are asymptomatic.

Diagnosis can be made by finding T. gondii in host tissue removed by biopsy or at necropsy. A rapid diagnosis may be made by making impression smears of lesions on glass slides. After drying for 10–30 minutes, the smears are fixed in methyl alcohol and stained with Giemsa. Well-preserved T. gondii are crescent-shaped and stain well with any of the Romanowsky stains (Fig. 47.2). In sections, the tachyzoites usually appear as oval to round and only half the size of those in smears (Fig. 47.11). Electron microscopy can aid diagnosis. Toxoplasma gondii tachyzoites are always located in vacuoles and have rhoptries with honeycomb structure (Fig. 47.3). Tissue cysts are without septa and with a thin cyst wall butted against the host cell plasmalemma (Figs. 47.4, 47.5). Occasionally, tissue cysts might be found in areas with lesions. The immunohistochemical staining of parasites with T. gondii antiserum can aid in diagnosis (Fig. 47.12).

Using secretions, excretions, body fluids taken by biopsy it is possible to search for T. gondii microscopically or for toxoplasmal DNA using of the polymerase chain reaction (PCR). The PCR and other gene amplification techniques can be designed to target specific regions of the parasite genome. The most commonly used gene of T. gondii is B1. By gene amplification, minute quantities of DNA, representing one organism, can be detected within a few hours. The main drawbacks of these methods are the inability to differentiate between dead and live organisms and the possibility of false positives originating during sample collection and processing. It is advisable not to entirely rely on this test for making vital decisions involving the fetus.

Diagnosis of T. gondii infection in the pregnant women, fetus, and the newborn is difficult and may require a combination of several serological tests to estimate the duration and onset of infection and this subject was recently reviewed (Remington et al. 2004, 2006; Petersen 2007; Petersen and Liesenfeld 2007).

Sulphadiazine and pyrimethamine (Daraprim^®) are two drugs widely used for therapy of toxoplasmosis. These two drugs act synergistically by blocking the metabolic pathway involving p-taminobenzoic acid and the folic-folinic acid cycle, respectively. These drugs are usually well tolerated but sometimes thrombocytopenia or leucopenia may develop. These effects can be overcome by administering folinic acid and yeast without interfering with treatment because the vertebrate host can utilize presynthesized folinic acid while T. gondii cannot (Frenkel 1973). While these drugs have a beneficial action when given in the acute stage of the disease process when there is active multiplication of the parasite, they will not usually eradicate infection. It is believed that these drugs have little effect on cysts. Sulfonamides are excreted within a few hours of administration; therefore, treatment has to be administered in daily divided doses (four doses of 500 mg each) usually for several weeks or months. A loading dose (75 mg) of pyrimethamine during the first 3 days has been recommended because it is absorbed slowly and binds to tissues. From the fourth day, the dose of pyrimethamine is reduced to 25 mg, and 2–10 mg of folinic acid and 5–10 g of bakers’ yeast are added (Frenkel 1973; St Georgiev 1994).

The plasma half-life of pyrimethamine is 35–139 hours. It has been administered in varying doses and schedules from daily to every 3 or 4 days. Pyrimethamine can cause nausea, headache, dysgeusia, thrombocytopaenia, and anaemia. Diagnosis and duration of treatment may have to be varied depending on the patient’s age and condition (Table 47.4). For example, prenatally infected children, whether with or without clinical manifestations, should be treated for at least one year.

Spiramycin, clindamycin, atovaquone, azithromycin, roxithromycin, clarithromycin, dapsone, and several other less commonly used drugs are available for treatment of toxoplasmosis and these were reviewed by McCabe (2001). Spiramycin is relatively nontoxic to mother and the fetus. It is concentrated in placenta and binds to tissues. Thus, it is used to prevent transmission of T. gondii from mother to the fetus.

Clindamycin is absorbed quickly and diffuses well into the central nervous system and therefore, has been used as alternative to sulfadiazine. It is rarely used to treat the primary maternal infection in pregnancy or congenital infection because it enters foetal blood when given to pregnant women. A major side effect of clindamycin is ulcerative colitis. McCabe (2001) has discussed in detail treatment of toxoplasmosis in patients with various clinical manifestations.

Before being given immunosuppressive treatments, patients should be serologically tested and, if devoid of T. gondii antibodies, be treated with pyrimethamine and sulfadiazine. This is particularly desirable in the case of patients receiving organ transplants. As it is usually impossible to carry out serologic tests on donors, they must all be considered potentially dangerous. There will, however, be time to test the recipients, and if seronegative, they should certainly be given prophylactic treatment. This strategy appears to have been effective in reducing the incidence of latent T. gondii infection in patients given marrow transplants from seronegative donors. Perhaps prophylactic treatment should be given to all recipients, irrespective of their sero-status.

Prophylactic treatment of all AIDS patients with T. gondii antibodies is desirable. Fortunately, some drugs used to prophylactically treat Pneumocystis pneumonia and bacterial infections may also prevent onset of clinical toxaplasmosis. Trimethoprim (160 mg) and sulfamethoxazole (800 mg) (twice daily and two times per week) combination is often used because it is inexpensive, convenient and works against Pneumocystis (McCabe 2001).

Cutaneous hypersensitivity, however, can be a problem and then alternative therapies are sought. A combination of dapsone (100 mg) and pyrimethamine (25 mg) orally weekly has also been used effectively. Other combinations of pyrimethamine and sulfanomides (Fansidar, three tablets every two weeks) have also been used.

Prevention of infection of the fetus by prophylactic treatment of the mother depends on the delay which occurs between maternal infection and its transmission to the fetus. It is also hoped that if infection is already present in the fetus, treatment may limit its ill effects. Serologic surveillance to detect maternal T. gondii infection during pregnancy is compulsory in Austria and France and is being applied to a few other countries. Treatment is initiated as soon as possible during the prenatal incubation period. In Austria, it is by spiramycin before the twentieth week of pregnancy and thereafter by pyrimethamine and sulfonamide, and in France it is by spiramycin alone. If these measures are begun sufficiently early, they may be expected to reduce the incidence of congenital toxoplasmosis by 50 to 70%.

In places where it has been carried out with thoroughness, persistence and determination, as in France, education appears to have contributed to a reduction in the incidence of T. gondii infection during pregnancy. Information regarding T. gondii infection should be included with the general instructions given in antenatal clinics and by obstetricians and midwives dealing with individual patients. Personal instruction given by word of mouth is likely to be most effective, and should be supplemented by booklets printed in various languages and by videos in the waiting rooms of antenatal clinics.

Treatment schedules are summarized in Table 47.4.

Toxoplasma gondii infection in humans is widespread and occurs throughout the world. Approximately one-half billion humans have antibodies to T. gondii. Infection rates in humans and others animals differ from one geographical area of a country to another. The causes of these variations are not yet known. Environmental conditions, cultural habits of the people, and animal fauna are some of the factors that may determine the level of infection with T. gondii. Infection is more prevalent in hot and humid areas than in dry and cold climates. Only a small proportion (less than 1%) of people acquire infection congenitally.

Women produce children with congenital infection generally once. Mothers of congenitally infected children have not been known to give birth to infected children in subsequent pregnancies.

The relative frequency of acquisition of postnatal toxoplasmosis due to eating raw meat and that due to ingestion of food contaminated by oocysts from cat faeces is not known and is difficult to investigate. Toxoplasma gondii infection is common in many animals used for food. Sheep, pigs, and rabbits are commonly infected throughout the world. Infection in cattle is less prevalent than in sheep or pigs. Infection is common in many species of wildlife, especially in deer and bears (Dubey 1994; Dubey and Jones 2008). Toxoplasma gondii tissue cysts survive in live food animals for years. As stated earlier, humans can acquire infection by eating raw or undercooked meat.

Toxoplasma gondii organisms in meat are susceptible to extremes of temperatures. Tissue cysts are killed by cooking meat to 67°C. Toxoplasma gondii in meat is killed by cooling to −13°C. Tissue cysts are also killed by exposure to 0.5 kGy of gamma irradiation.

Cultural habits of people may play a role in acquiring T. gondii infections. For example, in France the prevalence of Toxoplasma antibody is very high. The higher incidence in France appears to be related in part to the French habit of eating some of their meat raw. The high prevalence of T. gondii infection in Central and South America is in part due to high levels of contamination of the environment by oocysts (Dubey and Beattie 1988; Jones and Dubey 2010).

Oocysts are shed by domestic cats and wild felids. Widespread infection of the environment is possible because a cat may excrete millions of oocysts after ingesting one infected mouse. Oocysts are resistant to most ordinary environmental conditions and can survive in moist conditions for months and even years. Invertebrates, such as flies, cockroaches, dung beetles, and earthworms, can spread oocysts mechanically and even carry them onto food.

While only a few cats may be shedding T. gondii oocysts at any given time, the enormous numbers shed is important in the spread of T. gondii. Whether cats normally shed oocysts only once or several times during their lifetime is not known; however, under experimental conditions, cats develop good immunity to T. gondii against oocyst shedding but can reshed oocysts after re-inoculation of tissue cysts (Dubey 1995). Congenital infection can occur in cats and congenitally infected kittens can excrete oocyts. Infection rates of cats probably vary with the rate of infection in local avian and rodent populations because cats are thought to become infected in nature by eating these animals.

Theoretically, transmission of toxoplasmosis may be by sexual means, by ingestion of milk, saliva, or by eating of eggs. The stage most likely to be involved in these transmissions would be tachyzoites. Tachyzoites are delicate and do not survive outside the body for long. Therefore, there is practically no risk of transmission by kissing or by venereal transmission. There is little, if any, danger of T. gondii infection by drinking cow’s milk and, in any case, milk is generally pasteurized or even boiled. However infection has followed drinking unboiled goat’s milk. Raw hens’ eggs, although an important source of Salmonella infection, are extremely unlikely to transmit T. gondii infection.

Transmission by transplantation is also important (Wreghitt and Hakim 1989). Toxoplasmosis may arise in two ways in people undergoing transplantation: from implantation of an organ or bone marrow from an infected donor into a non-immune immunocompromised recipient, and from induction of disease in an immunocompromised latently infected recipient. In the later case, the immunosuppressive treatment activates the latent infection of the recipient. In these cases both tachyzoites and tissue cysts might be involved, but more probably tissue cysts. In both cases the cytotoxic and immunosuppressive therapy given to the recipient is the cause of induction of the active infection and the disease.

The objectives of use of vaccines against toxoplasmosis include reducing fetal damage, reducing the number of T. gondii tissue cysts in animals, and preventing the formation of oocysts in cats (Araujo 1994; Dubey 1994). All of these objectives are not currently feasible with the use of a single vaccine. At present there are no effective subunit or killed vaccines for immunization against T. gondii but research is under way in many laboratories.

One vaccine that contains a strain (S48) of tachyzoites that does not persist in the tissues of sheep is available in Europe and New Zealand to reduce fetal losses attributable to toxoplasmosis (Buxton 1993). Ewes vaccinated with the S48 strain vaccine retain immunity for at least 18 months (Buxton 1993). However, this vaccine does not prevent reinfection and encystment of wild strain of T. gondii.

To prevent infection of human beings by T. gondii, hands should be washed thoroughly with soap and water after handling meat. All cutting boards, sink tops, knives, and other materials coming in contact with uncooked meat should be washed with soap and water. This is effective because the stages of T. gondii in meat are killed by soap and water. Meat of any animal should be cooked to 67°C before consumption, and tasting meat while cooking or seasoning home-made sausages should be avoided. Pregnant women, especially, should avoid contact with cats, soil, and raw meat. Pet cats should be fed only dry, canned, or cooked food. The cat litter box should be emptied every day, preferably not by a pregnant woman. Gloves should be worn while gardening. Vegetables should be washed thoroughly before eating because they may have been contaminated with cat faeces. Expectant mothers should be aware of the dangers of toxoplasmosis.

To prevent infection in cats, they should never be fed uncooked meat, viscera, or bones, and efforts should be made to keep cats indoors to prevent hunting. Trash cans also should be covered to prevent scavenging.

Cats should be neutered to control the feline population on farms. Dead animals should be removed promptly to prevent cannibalism by pigs and scavenging by cats. Sheep that have aborted due to toxoplasmosis usually do not have subsequent toxoplasmic abortions, and thus can be saved for future breeding. Fetal membranes and dead fetuses should be not be handled with bare hands and should be buried or incinerated to prevent infection of felids and other animals on the farm. Cats should not be allowed near pregnant sheep and goats. Grain should be kept covered to prevent oocyst contamination.

To prevent infection of zoo animals with T. gondii, cats, including all wild Felidae, should be housed in a building separate from other animals, particularly marsupials and New World monkeys. Cats as a rule should not be fed uncooked meat. However, if a choice has to be made, frozen meat is less likely to contain live T. gondii than fresh meat, and beef is less likely to contain T. gondii than is horse meat, pork, or mutton. Dissemination of T. gondii oocysts in the zoo should be prevented because of potential exposure of children. Brooms, shovels, and other equipment used to clean cat cages, and cat enclosures should be autoclaved or heated to 67°C for at least 10 minutes at regular intervals. While cleaning cages, animal caretakers should wear masks and protective clothing. Feline faeces should be removed daily to prevent sporulation of oocysts.

The Sarcocystis parasite was first found in the skeletal muscle of a house mouse, Mus musculus in Switzerland in 1843 (Table 47.5). Before 1972, many of these parasites were named based upon the finding of cysts in the muscles of a host. The true nature of these intramuscular cysts remained unknown until the discovery of the life cycle of Sarcocystis in 1972 (Table 47.5).

Sarcocystis species are coccidian parasites, classified in the family Sarcocystidae (Poche 1913), subfamily, Sarcocystinae (Poche 1913), and genus, Sarcocystis (Lankester 1882).

Sarcocysts (in Greek sarkos = flesh, kystis = bladder) are the terminal asexual stage of development of these parasites. They are found primarily in the striated muscles of mammals, including humans (Fig. 47.16), birds, marsupials, and poikilothermic animals.

Sarcocystis has an obligatory prey-predator two host life cycle (Fig. 47.17). Asexual stages develop only in the intermediate host, which in nature is often a prey animal. Sexual stages develop only in the definitive host, which is carnivorous.

The intermediate host becomes infected by ingesting sporocysts in food or water. Sporozoites excyst from sporocysts in the small intestine and produce intravascular meronts that give rise to the encysted form (sarcocyst) in muscles. Sarcocysts become infectious only when they contain bradyzoites (Dubey 2005c).

The definitive host become infected by ingesting tissues containing mature sarcocysts. Bradyzoites liberated from the sarcocyst by digestion in the stomach and intestine transform into male (micro) and female (macro) gamonts. After fertilization of macrogamete by microgamete, a wall develops around the zygote and the oocyst is formed. The entire process of gametogony and fertilization can be completed within 24 hours, and gamonts and oocysts may be found at the same time. Sarcocystis species oocysts sporulate in the lamina propria (Fig. 47.18). Sporulated oocysts are generally colourless, thin-walled (< 1 μm), and contain two elongated sporocysts. Each sporocyst contains four elongated sporozoites and a residual body. The oocyst wall is thin and often ruptures. Free sporocysts, released into the intestinal lumen, are passed in the faeces.

Unlike Toxoplasma, there are more than 100 species of this genus (Dubey et al. 1989). Only certain species of Sarcocystis are pathogenic to intermediate hosts (Dubey et al. 1989). Generally species transmitted by canids are more pathogenic than those transmitted by felids. Sarcocystis generally does not cause illness in definitive hosts.

Sarcocystis neurona is an unusual species of the genus that does not follow the lifecycle pattern of other species outlined above. It is also one of the most pathogenic species of the genus. Sarcocystic neurona is the most frequent cause of a fatal equine protozoal encephalomyelitis (EPM) in horses in the Americas (Dubey et al. 2001). Horses are considered the aberant host because only schizonts are found in their tissues. Unlike other species, S. neurona schizonts occur in neural cells, not in the vascular endothelium. Its sarcocysts occur in domestic cats, striped skunks, raccoons, sea otters, and armadillos. Opossums (Didelphis virginianus, D. abbreventis) are its definitive hosts. Only the sexual cycle occurs in the definitive host and it is confined to the small intestine. Encephalomyelitis associated with S. neurona has been reported in horses, ponies, zebras, skunks, raccoons, cats, lynx, mink and marine mammals (Pacific harbour seals and sea otters).

Sarcocystis canis is another unusual species of the genus with an unknown life cycle. Its sarcocysts, sexual phase and definitive hosts are unknown. Schizont is the only stage that is known. Sarcocystis canis has been found associated with fatal hepatitis in sea lions, dogs, black and grizzly bears, a horse, and a dolphin (Dubey and Speer 1991; Dubey et al. 2006).

There are two known species of Sarcocystis for which humans serve as the definitive host, S. hominis and S. suihominis (

Murrell et al. 1985). Humans also serve as accidental intermediate hosts for several unidentified species of Sarcocystis. Symptoms in persons with intestinal sarcocystosis are different from those persons with muscular sarcocystosis and vary with the species of Sarcocystis causing infection.

Infection with this species is acquired by ingesting uncooked beef containing S. hominis sarcocysts. Sarcocystis hominis is only mildly pathogenic for humans. Volunteers who ate raw beef developed nausea, stomach ache, and diarrhoea 3–6 hours after ingesting the beef; these symptoms lasted 24–36 hours. The volunteers excreted S. hominis sporocysts between 14 and 18 days after ingesting the beef (Aryeetey and Piekarski 1976; Heydorn 1977). In one report, six of seven human volunteers that ate 128–260 g of Kibbe (a preparation made from raw beef and spices) obtained from an Arabian restaurant in Saõ Paulo, Brazil, excreted S. hominis sporocysts 10–14 days later. Two of these volunteers became ill. One of them had abdominal pain and diarrhoea 1–3 days p.i. and the other had diarrhoea 11 days p.i. (Pena et al. 2001). A patient in Spain developed abdominal pain and loose stools after eating raw beef; the diagnosis was confirmed by finding S. hominis sporocysts in faeces (Clavel et al. 2001).

A Sarcocystis species similar to S. hominis named S. dubeyi (Huong and Uggla 1999) has been found in water buffaloes. The definitive host for S. dubeyi is unknown but suspected to be humans.

This species, acquired by eating undercooked pork, is more pathogenic than S. hominis. Human volunteers developed hypersensitivity-like symptoms; nausea, vomiting, stomach ache, diarrhoea, and dyspnoea within 24 hours of ingestion of uncooked pork from naturally or experimentally infected pigs. Sporocysts were shed 11–13 days after ingesting pork (Rommel and Heydorn 1972; Piekarski et al. 1978; Hiepe et al. 1979; Kimmig et al. 1979; Dubey et al. 1989). Zho (1991) found sporocysts in stools of 123 of 414 (29.7%) persons from 3 villages in Xianguan City in China. He reported that these people ate raw pork but not raw beef.

Before the discovery of the life cycle of Sarcocystis and recognition of cattle and pigs as sources of human infection, Sarcocystis sporocysts in human faeces were referred to as Isospora hominis. Because of structural similarities between S. hominis and S. suihominis sporocysts, it is not possible to distinguish between them by microscopic examination. Intestinal sarcocystosis is more common in Europe than in other continents. Enteritis was associated with shedding of Sarcocystis sporocysts in some cases (reviewed in Dubey et al. 1989).

Sarcocysts have been found in striated muscles of human beings, mostly as incidental findings. Judging from the published reports, sarcocysts in humans are rare (Beaver et al. 1979; Dubey et al. 1989). Most reported cases were from Asia and south east Asia. Sarcocysts were found in both in skeletal and cardiac muscles. The clinical significance of sarcocysts and their life cycles in humans are unknown. In one report, 7 of 15 USA military men developed acute illness after an army exercise in rural Malaysia (Arness et al. 1999). The illness was characterized by fever, myalgias, bronchospasm, fleeting pruritic rashes, transient lymphadenopathy, and subcutaneous nodules associated with eosinophilia, elevated erythrocyte sedimentation rate, and elevated levels of muscle creatinine kinase. Sarcocysts of an unidentified Sarcocystis species were found in skeletal muscle biopsies of the index case. Symptoms in five other men were mild to moderate and self-limited, and one team member with laboratory abnormalities was asymptomatic. Of eight team members tested for antibody to Sarcocystis, six were positive; of four with the eosinophilic myositis syndrome who were tested, all were positive. The illness was considered to be sarcocystosis. Arness et al. (1999) also reviewed other cases of sarcocystosis after 1990.

Poor hygiene practiced in underdeveloped countries during handling of meat from slaughter place to kitchen can be a source of Sarcocystis infection. In one survey in India, S. suihominis oocysts were found in the faeces of 14 out of 20 three to twelve year old children (Banerjee et al. 1994), indicating that meat was consumed raw at least by some because S. suihominis can only be transmitted to humans by the consumption of raw pork. In another study, 3–5 year old children from a slum area were found to consume meat scraps virtually raw, and many pigs from that area harboured S. suihominis sarcocysts (Solanki et al. 1991). In European countries where consumption of raw or undercooked meat is relatively high, humans are expected to have intestinal sarcocystosis (Dubey et al. 1989). To prevent intestinal infection, meat should be cooked thoroughly before human consumption.

The ante-mortem diagnosis of muscular sarcocystosis can only be made at present by histological examination of muscle collected by biopsy. The finding of immature sarcocysts with metrocytes suggests recently acquired infection. The finding of mature sarcocysts only indicates past infection. The diagnosis of intestinal sarcocystosis is easily made by faecal examination. As said earlier, sporocysts or oocysts are shed fully sporulated in faeces. It is not possible to distinguish species of Sarcocystis based on sporocyst morphology.

There is no treatment known for Sarcocystis infections of humans. The intestinal phase of Sarcocystis is caused by oocysts in the lamina propria of small intestine; there are no drugs that can kill oocysts in situ. The muscular phase is rarely diagnosed ante-mortem.

Isospora belli (Wenyon 1923), is the cause of coccidiosis in humans. It belongs to the family Eimeriidae and the genus, Eimeria. Most of the reported cases occurred in the tropics rather than in the temperate zone. Infection is now seen more frequently in immunocomprised patients, particularly those with AIDS (Restrepo et al. 1987; Dubey 1993; Michiels et al. 1994; Lindasy et al. 1997; Velàsquez et al. 2001; Jongwutiwes et al. 2007; Karanis et al. 2007).

Isospora belli oocysts are elongate, ellipsoidal, and are 20–33 × 10–19 μm (Fig. 46.19A). Sporulated oocysts contain two ellipsoidal sporocysts without a Stieda body. Each sporocyst is 9–14 × 7–12 μm and contains four crescent-shaped sporozoites and a residual body. Sporulation occurs within 5 days, both within the host and in the external environment (Trier et al. 1974). Thus, both unsporulated and sporulated oocysts may be shed in faeces.

Infection occurs by the ingestion of food contaminated by oocysts. Merogony and gametogony occur in the upper small intestinal epithelial cells, from the level of the crypts to the tips of the villi. The number of generations of merogony is unknown. In AIDS patients the parasite may be disseminated to extraintestinal organs, including mesenteric and mediastinal lymph nodes, liver, and spleen (Michiels et al. 1994). Single zoites surrounded by a capsule (cyst wall) have a prominent refractile or crystalloid body, indicating that the encysted organisms are sporozoites (Lindsay et al. 1997). Organisms with a cyst wall are found only in extra intestinal organs.

Isospom belli can cause severe clinical symptoms with an acute onset, particularly in AIDS patients. Infection has been reported to cause fever, malaise, cholecystitis, persistent diarrhoea, weight loss, steatorrhoea, and even death (Dubey 1993).

Diagnosis can be established by finding characteristic bell-shaped oocysts in the faeces (Fig. 47.19A) or coccidian stages in intestinal biopsy material (Dubey 1977). Affected intestinal portions may have a flat mucosa, similar to that occurring in sprue. The stools during infection are fatty and at times very watery.

Sulphonamides are considered effective against coccidiosis (St Georgiev 1993; Lindsay et al. 1997). Treatment with TMP-SMX (trimethoprim-sulfa) has been used most often.

Cyclospora cayetanensis (Ortega et al. 1994) is the cause of disease in humans. Historically, the parasite was probably first recognized by Ashford (1979) in faeces of patients in Papua New Guinea (Herwaldt 2006). Ortega et al. (1994) named it Cyclospora cayetanensis.

Cyclospom cayetanensis oocysts are approximately 8 μm in diameter and they contain two ovoid 4 × 6 μm sporocysts (Fig. 47.19B,C). Each sporocyst has two sporozoites. Thus there are a total of four sporozoites in a sporulated oocyst. Unsporulated oocysts are excreted in faeces. Sporulation occurs outside the body, sometimes after several weeks. Other stages in the life cycle are not known. Transmission is thought to be by the ingestion of food and water contaminated with sporulated oocysts. Several clinical outbreaks have been reported in humans in USA and Canada epidemiologically linked to ingestion of imported fresh produce such as raspberries, snow peas, and mesculun lettuce (Herwarldt 2006).

Both immunocompetent and immunosuppressed patients of all ages may have coccidia, fever, nausea, chills, dizziness, fatigue, and abdominal cramps. Infection has been reported from several countries (Adal 1994; Ortega et al. 1993, 1994; Herwarldt, 2006; Ortega and Sanchez 2010).

Diagnosis can be made by faecal examination. Cyclospora oocysts are approximately 8 μm, remarkably uniform, and contain a sporont (inner mass) that occupies most of the oocyst. They are acid-fast, autofluorscent, and need to be distinguished from Cryptosporidium oocysts which are not autofluorescent. Unlike cryptosporidial oocysts, C. cayetanensis oocysts have a much thicker oocyst wall and their contents are more granular than are those of cryptosporidial oocysts.

Successful treatment has been reported with trimethoprim-sulfamethazole or nitazonamide (Verdier et al. 2000).