Scholarly article on topic 'Foodborne and waterborne zoonotic sarcocystosis'

Foodborne and waterborne zoonotic sarcocystosis Academic research paper on "Biological sciences"

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Food and Waterborne Parasitology
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{" Sarcocystis hominis " / " Sarcocystis heydorni " / " Sarcocystis suihominis " / " Sarcocystis nesbitti "}

Abstract of research paper on Biological sciences, author of scientific article — J.P. Dubey

Abstract The ingestion of raw beef or pork infected with Sarcocystis can cause illness in humans. Nausea and vomiting can occur within three days of consuming infected meat; these symptoms are considered due to toxic substances in sarcocysts or to other factors in raw meat. Diarrhea and abdominal pain are associated with excretion of sporulated sporocysts in human feces usually within 8–14days after ingestion of infected meat. The intestinal phase is often self-limiting but sporocysts may be excreted for months. There appears to be little or no immunity to excretion of sporocysts after ingesting each infected meal. The clinical illness is more severe after eating infected pork versus infected beef. There is one zoonotic Sarcocystis species in pork, Sarcocystis suihominis, with domestic and wild pigs as intermediate hosts. Two zoonotic species are present in cattle, Sarcocystis hominis, and Sarcocystis heydorni; neither has been recognized in North America. Although isolated human cases of muscular sarcocystosis have been known for more than 100years, recently a mysterious serious, diagnostically challenging illness has been reported in humans on vacation/travel to Malaysia; sarcocysts were detected histologically in biopsy of muscles from some of these patients. These outbreaks of sarcocystosis-like illness in humans are summarized. Molecular epidemiological evidence suggests a new type of zoonosis linked to ingestion of food and water contaminated with “Sarcocystis nesbitti”. The life cycle of S. nesbitti remains unknown. Maccaque monkeys are thought to be its intermediate hosts and unknown species of snakes as definitive hosts. Evidence possibly linking S. nesbitti to human outbreaks or lack of it is reviewed.

Academic research paper on topic "Foodborne and waterborne zoonotic sarcocystosis"

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Food and Waterborne Parasitology

journal homepage: www.elsevier.com/locate/fawpar

Foodborne and waterborne zoonotic sarcocystosis

J.P. Dubey *

United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA

ARTICLE INFO ABSTRACT

The ingestion of raw beef or pork infected with Sarcocystis can cause illness in humans. Nausea and vomiting can occur within three days of consuming infected meat; these symptoms are considered due to toxic substances in sarcocysts or to other factors in raw meat. Diarrhea and abdominal pain are associated with excretion of sporulated sporocysts in human feces usually within 8-14 days after ingestion of infected meat. The intestinal phase is often self-limiting but sporocysts may be excreted for months. There appears to be little or no immunity to excretion of sporocysts after ingesting each infected meal. The clinical illness is more severe after eating infected pork versus infected beef. There is one zoonotic Sarcocystis species in pork, Sarcocystis suihominis, with domestic and wild pigs as intermediate hosts. Two zoonotic species are present in cattle, Sarcocystis hominis, and Sarcocystis heydorni; neither has been recognized in North America. Although isolated human cases of muscular sarcocystosis have been known for more than 100 years, recently a mysterious serious, diagnostically challenging illness has been reported in humans on vacation/travel to Malaysia; sarcocysts were detected histologically in biopsy of muscles from some of these patients. These outbreaks of sarcocystosis-like illness in humans are summarized. Molecular epidemiological evidence suggests a new type of zoonosis linked to ingestion of food and water contaminated with "Sarcocystis nesbitti". The life cycle of S. nesbitti remains unknown. Maccaque monkeys are thought to be its intermediate hosts and unknown species of snakes as definitive hosts. Evidence possibly linking S. nesbitti to human outbreaks or lack of it is reviewed.

Published by Elsevier Inc.

CrossMark

Article history:

Received 7 August 2015

Received in revised form 2 September 2015

Accepted 4 September 2015

Available online 8 October 2015

Keywords: Sarcocystis hominis Sarcocystis heydorni Sarcocystis suihominis Sarcocystis nesbitti

Contents

1. Introduction to general biology of Sarcocystis......................................... 3

2. Sarcocystis as agent of zoonosis............................................... 3

2.1. Humans as definitive hosts.............................................. 3

2.1.1. Zoonotic species of Sarcocystis infecting pigs ................................. 5

2.1.2. Zoonotic species of Sarcocystis infecting cattle................................. 6

2.2. Humans as intermediate hosts of Sarcocystis...................................... 6

2.2.1. Isolated cases............................................... 6

2.2.2. Outbreaks of mysterious illnesses clinically and epidemiologically associated with Sarcocystis........... 6

Conflict of interest............................................................................................................10

Acknowledgments............................................................................................................10

References....................................................................................................................10

* Tel.: +1 301 504 8128; fax: +1 301 504 9222. E-mail address: Jitender.dubey@ars.usda.gov.

http://dx.doi.org/10.10167j.fawpar.2015.09.001 2405-6766/Published by Elsevier Inc.

1. Introduction to general biology of Sarcocystis

Sarcocystis species (Apicomplexa: Sarcocystidae) are coccidian parasites of warm blooded and poikilothermic animals, including humans. More than 200 species of Sarcocystis are recognized however, complete life cycles are known for only 26 species (Dubey et al., 2015a). Sarcocystis species usually have a strict two-host cycle with herbivores as intermediate hosts and carnivores as definitive hosts. The carnivore host becomes infected by ingesting tissues of herbivores with encysted stage (sarcocyst) of the parasite containing hundreds of bradyzoites. The bradyzoites transform into male and female gamonts in the small intestine within a day of ingestion of the infected meat; there is no multiplication of the parasite in the carnivore host. Gametogony usually occurs in the lamina propria of the small intestine, leading to the formation of unsporulated oocysts (Fig. 1). The oocysts sporulate in situ, usually within one week after ingestion of bradyzoites. The oocyst wall is fragile and breaks releasing two sporocysts. Each sporocyst contains four sporozoites. Because the oocysts/sporocysts are trapped in the lamina propria, their release into the intestinal lumen and feces endures for several months. Sporocysts are the environmentally resistant stage.

The herbivore host acquires infection by ingesting feed and water contaminated with Sarcocystis sporocysts. After sporocysts are ingested, sporozoites are liberated from sporocysts, and initiate a complex asexual cycle. The sporozoite migrates from the intestine to extra-intestinal tissues. The sporozoite nucleus divides into many lobes, eventually forming merozoites. The multilobed dividing stage is called schizont. The schizonts are formed in a variety of cells, including vascular endothelial cell, somatic cells, and neuronal cells, depending on the species of Sarcocystis. After a few cycles of schizonts, the parasite encysts, usually in a myocyte, first forming metrocytes that give rise to bradyzoites. The encysted stage is called a sarcocyst. A sarcocyst may take a month or more to mature and become infectious for the carnivore host. Mature sarcocysts can contain numerous bradyzoites, and the sarcocysts may grow in size for years to become macroscopic. Only mature sarcocysts containing bradyzoites are infectious for the carnivore host. The bradyzoites are not infectious for the intermediate host and sporocysts are not infectious for the definitive host. Thus, the parasite has a strict 2-host life cycle. With few known exceptions, Sarcocystis species are host-specific. The biology of Sarcocystis species has been reviewed in detail recently by Dubey et al. (2015a).

The purpose of the present paper is to summarize information on zoonotic species of Sarcocystis.

2. Sarcocystis as agent of zoonosis

2.1. Humans as definitive hosts

Until the discovery of the two-host life cycle of Sarcocystis in 1972, fully sporulated oocysts or sporocysts in human feces were called Isospora hominis. It was subsequently recognized that these sporulated oocysts were most probably Sarcocystis. Of all the mammals, birds, and poikilothermic hosts infected with Sarcocystis species, only cattle and pigs are confirmed intermediate hosts of zoonotic Sarcocystis species (Dubey et al., 2015a).

Human volunteers excreted fully sporulated oocysts or sporocysts in feces eight or more days after ingesting raw beef or pork infected with Sarcocystis (Table 1). Some of them became ill, depending on the type of meat (beef or pork) ingested and the intensity of sarcocysts in the meat. The people who ate pork had more serious symptoms than those who ate beef. Symptoms within three days of ingestion of raw meat are probably related to toxins (Dubey et al., 2015a) released from invading bradyzoites in raw meat (Table 1). Diarrhea and abdominal pain were among the most consistent symptoms following sporocyst

Human eats undercooked meat containing mature sarcocysts

Unsporulated oocyst

Sporulated oocyst

Fig. 1. Life cycle of two Sarcocystis species with humans as definitive hosts. (From Fayer et al., Clin. Microbiol. Rev. 28:295-311, 2015).

Table 1

Patent Sarcocystis spp. infection in humans who ate raw/undercooked meat.

Meat Country Source No. of persons Symptoms Sporocysts in feces Reference

(prepatent period)

Beef Germany Naturally infected 2 No symptoms Yes (9) patent Rommel and

500 g All ingested raw meat regularly period at least Heydorn (1972)

Turkey Naturally infected 1a 14.11.1973 Dizziness, nausea, abdominal pain 40 d.p.i. Yes (10) Heydorn (1977)

Germany Experimentalb Dr. Heydorn 3-6 h later, and diarrhea 24-36 h

98 days p.i. 30.04.1974 later. Diarrhea and stomachache later

6 months p.i 29.07.1974 during sporocyst excretion

4 months p.i. 18.05.1975

Germany Naturally infected 2 1 person showed no symptoms. The Yes (9-12) Hiepe et al.

100 g (100 cysts other have stomachache, nausea and (1979)

aproximately) diarrhea 5-7 dpi.

Germany Naturally infected 12 No symptoms Yes 5 of 12 Aryeetey and

300-500 g (13-39) Piekarski (1976)

(100 g/day during Patent period

3-5 days) between 9 and

1 person intake 179 d.p.i.

other 2 times

(total 3 infections)

Brazil Naturally Infected 7 6 volunteers excreted sporocysts. 2 Yes (10-14) Pena et al.

(kibbe from had symptoms. One person had (2001)

Arabian food nausea and abdominal pain on day

stores) 1 p.i., and diarrhea for the first 3 days.

The second person had diarrhea on

day 11 p.i.

China Experimentally 1 Abdominal pain, diarrhea, fatigue, Yes (8) Lian et al.

lnfected-150 d.p.i. dizziness on day 3 p.i. (1990)

Pork Germany Naturally infected 4 Only 1 person showed diarrhea and Yes 3 of 4 persons Rommel and

125 g All ingested regularly raw meat fever. (10-13) patent Heydorn (1972)

period at least

30 d.p.i.

Germany Naturally infected 3 (1st intake 400 g mild infected, the Anorexia, bloating, nausea, Yes (9 d.p.i, Heydorn (1977)

150-400g 2nd intake 150-200 g and further stomachache, dry heaves, diarrhea reported only for 1

50 g at 5 days heavily infected, and 6-8 h later, and lasting up to 48 h. of the 3 persons)

the 3rd intake 200 g heavily infected)

Germany Experimentalc 8 (additional 4 persons were Diarrhea, stomachache, vomiting, Yes (11-13) Piekarski et al.

142 d.p.i.; 100- uninfected control) sweats 6-24 h later. Also abdominal patent period at (1978)

150 g and muscular pain and potentially least 71 d.p.i.

bloody faces (red stain). All

symptoms lasted 12-24 h. Three

weeks later diarrhea or fluid stools

which lasted 1 -2 weeks.

Germany Experimentald 14(3 were uninfected control) Diarrhea, stomachache, headache and Yes (at 3 weeks Kimmig et al.

175 d.p.i.; 20- nausea, related to amount of infected p.i. all were (1979)

70 g. pork ingested. Symptoms appear positive as well as

4-16 h p.i. and last 14 h to 3.5 d.p.i. 5 weeks p.i.)

China Experimental 1 None Yes (12) Li and Lian

144 d.p.i. (1986)

d.p.i = days post ingestion.

a Dr. Heydorn ate 400 g raw minced beef over a period of 3 consecutive days (200,100 and 100 g/day). Beef was from Ankara, Turkey. The beef was heavily infected with thick and thin walled sarcocysts.

b For experimental infection of cattle with S. hominis sporocysts see Gestrich et al. (1975). Five calves (#1-5) were fed 420,000 (calves#1,2), 2,000,000 (calves #3-5) and they examined 62,98,48,48,188 days p.i. Sarcocyst development in calves was described by Heydorn et al. (1975) and Mehlhorn et al. (1975). The sporocysts used to inoculate cattle were derived from feces of Dr. Heydorn.

c Experimental infection: 1 pig was orally infected with 1 million sporocysts of S. suihominis and euthanized 142 dpi. Muscles samples were analyzed by trichinoscopy and bacteriological studies to discard other pathogens. Humans intake 100-150 g pork each (one day after the pig was slaughtered).

d Experimental infection: 1 pig was orally infected with 500,000 sporocysts of S. suihominis and euthanized 175 dpi. Muscles samples were analyzed by trichinoscopy and bacteriological studies to discard other pathogens. The persons intake 20-70 g pork each (one day after the pig was slaughtered). The volunteers were divided into 5 groups (G): G1: n = 4, intake 20 g pork each; G2: n = 4, intake 40 g pork each; G3: n = 3, intake 60 g pork each; G4: n = 3, intake 70 g pork each and G5: n = 3,as control group. Additionally, other 2 volunteers intake 80 g of pork which was frozen 24 h at -30 °C (no symptoms or sporocysts were detected in these persons).

excretion. Sarcocystis hominis sporocysts were 14.7 x 9.3 (13.1-17.0 x 7.7-10.8; n = 100) |jm and S. suihominis sporocysts were 12.6 x 9.3 (10.8 x —13.9 x 7.7-10.8; n = 133) |am in size (Rommel and Heydorn, 1972). Because of the overlap in sizes of spo-rocysts, S. hominis sporocysts cannot be reliably distinguished from S. suihominis sporocysts.

In addition to the reports listed in Table 1, there are numerous reports of findings of Sarcocystis sporocysts in human feces, mostly from Europe. We have recently summarized all of the published reports (Fayer et al., 2015).

Most of the volunteers listed in Table 1 were authors of the cited papers. It is noteworthy that one of these human volunteers was Dr. Heydorn who ate both infected beef and pork and carefully recorded sporocyst excretion, and was the source of inocula for experimental infections in cattle and pigs. For many years he maintained the beef-human and pork-human cycles in his laboratory. In addition to historical importance, the data are of epidemiological significance. Sporocysts were detected in feces after the ingestion of each infected meal, irrespective whether infected beef or pork was ingested, indicating that there is evidence against immunity preventing repeated sporocyst excretion, irrespective of the species of Sarcocystis ingested (Heydorn, 1977; personal communication to JPD July, 2015).

2.1.1. Zoonotic species of Sarcocystis infecting pigs

Pigs are listed as intermediate hosts for 3 Sarcocystis species: Sarcocystis miescheriana, Sarcocystis suihominis, and Sarcocystis porcifelis. Full details of the life cycle of S. miescheriana and S. suihominis are known (Dubey et al., 2015a). S. miescheriana and S. suihominis sarcocysts can also be distinguished molecularly, and morphologically (Dubey et al., 2015a) (Fig. 2). The taxonomic status of S. porcifelis is uncertain and its morphologic description is vague (Dubey et al., 2015a). Of these 3 species, only S. suihominis is zoonotic (Dubey et al., 2015a).

Sarcocystis miescheriana sarcocysts are up to 1500 |jm long and 200 |jm wide (Fig. 2 A, and C). The sarcocyst wall is 3 to 6 |jm thick and appears radially striated. The villar protrusions (vp) on the sarcocyst wall are up to 5 |jm long and 1.3 |am wide, type 10b (Dubey et al., 2015a).

S. suihominis has been reported in pigs from Europe, India, and Japan. Humans and nonhuman primates (Macaca mulatta, Macaca irus, Pan troglodytes, and Papio cynocephalus) are its definitive hosts. Its sarcocysts are up to 1500 |jm long. The sarcocyst wall is 4 to 9 |jm thick and appears hirsute, with vp up to 13 |jm long, type 31 (Dubey et al., 2015a) (Fig. 2B and D). Sarcocysts mature in 2 months.

Fig. 2. Sarcocysts of S. miescheriana (A and C) and S. suihominis (B and D) in skeletal muscles of pigs. The villar protrusions of S. suihominis are thinner and longer than those of S. miescheriana. A and C, Toluidine blue stain. C and D, TEM (From Dubey et al., 2015a).

2.1.2. Zoonotic species of Sarcocystis infecting cattle

Of the five species of Sarcocystis in cattle (S. cruzi, S. hirsuta, S. hominis, S. rommeli, and S. heydorni) only S. hominis and S. heydorni (Dubey et al., 2015b, 2015c) are zoonotic (Fig. 3). They can be distinguished morphologically (Table 2). Sarcocysts of S. cruzi and S. heydorni are thin-walled and they appear structurally similar under the light microscope, whereas sarcocysts of S. hirsuta, S. rommeli, and S. hominis are thick-walled and they are also difficult to speciate based on light microscopy (Fig. 3). These species can be distinguished by transmission electron microscopic structures of cyst walls (Fig. 4).

To my knowledge, only sarcocysts of S. hirsuta can become macroscopic. The biologic features of the five bovine species of sarcocysts are summarized in Table 2.

22. Humans as intermediate hosts of Sarcocystis

22.1. Isolated cases

During the last 112 years there have been many reports of finding sarcocysts in sections of muscles obtained by biopsy, or at post mortem in around 100 people; these were recently tabulated and summarized (Fayer et al., 2015). To this list a recent report from India should be added (Lingappa et al., 2015). The most significant of these reports is a study by Wong and Pathmanathan (1992) in Malaysia. They microscopically examined ten 5-^m histological sections from each of 100 humans older than 12 years. No selection was made based on symptoms or the cause for post mortem; most of them were victims of automobile accidents from July to December, 1990. Sarcocysts were found in tongues of 21 individuals; in half of these, only one sarcocyst was seen in 10 paraffin blocks of tongue cut at 1 to 2 cm apart. ln one person, 13 sarcocysts were found in 10 blocks. Thus, the density of sarcocysts in asymptomatic general human population in Kuala Lumpur was low. This report also suggests that one or more species of Sarcocystis can form mature sarcocysts in humans in Malaysia.

Most of these reports of sarcocysts in muscles were in patients from South East Asia, both in males and females, and in young (9 year old) and old (67 year old) (Fayer et al., 2015). To my knowledge, sarcocysts have not been reported from humans in the USA or Canada, excluding patients that were born and raised elsewhere or had traveled to Malaysia (see Table 3). A review of the literature revealed that several morphologically distinct, mature sarcocysts were present in histologically confirmed cases, and associated with a variety of symptoms (Beaver et al., 1979; Dubey et al., 2015a).

2.2.2. Outbreaks of mysterious illnesses clinically and epidemic/logically associated with Sarcocystis

Three mysterious, diagnostically challenging outbreaks of human illness had been reported in persons who had history of travel to Malaysia (central peninsular Malaysia in 1993, Tioman Island in 2011-2014, and Pangkor Island in early 2012), thought to be associated with a S. nesbitti-like parasite (Table 3; for reviews see Poulsen and Stensvold, 2014; Fayer et al., 2015; Harris et al., 2015). In the two recent outbreaks, most affected persons complained of fatigue, fever, and muscle weakness within three weeks of return from vacation/travel to Malaysia. Extensive laboratory investigations revealed blood eosinophilia, elevated liver and muscle enzymes (Esposito et al., 2014; Italiano et al., 2014). Testing for various viruses, bacteria, and helminths including Trichinella was negative. Because symptoms resembled aspects of toxoplasmosis, extensive testing was done but failed to reveal association with Toxoplasma gondii infection. Muscle biopsies on some of the patients revealed sarcocysts (Fig. 5), and PCR testing pointed to S. nesbitti infection (see later section). In the past, except for some cases of toxoplasmosis and Dientamoeba fragilis infection, blood eosinophilia was not a feature of protozoal infections. Some of the patients were severely ill and hospitalized. To

Fig. 3. Sarcocyst walls of five species of Sarcocystis in histological sections of cattle. A-C, hematoxylin and eosin stain, D, Periodic acid Schiff-hematoxylin, E, Tolu-idine blue stain. (A), S. cruzi, tongue, 98 days p.i. (B) = S. heydorni, tongue, 111 days p.i. (C) = S. hominis, tongue, 111 days p.i. (D), S. hirsuta, tongue, 110 days p.i. (E) = S. rommeli loin, naturally infected.

Table 2

Summary of biology of Sarcocystis species in cattle.

Character S. cruzi S. heydorni S. hominis S. hirsuta S. rommeli

Definitive hosts Schizonts Dogs and other canids Vascular endothelium of several organs Humans Unknown Humans Unknown Domestic cat Capillaries of striated muscle and heart Unknown Unknown

Sarcocysts Size Wall (LM) (TEM) typea Microscopic Thin 7a Microscopic Thin 29 Microscopic Thick 10b Macroscopic Thick 28 Microscopic Thick 10c

Tissue Skeletal muscle Heart Prevalence Yes Yes Worldwide Yes No Germany, Turkey Yes Rare Probably worldwide, not reported from USA and Canada Yes No Worldwide Yes Yes Argentina, Europe

LM = light microscopy. TEM = transmission electron microscopy. a Dubey etal. (2015a).

illustrate the seriousness of the illness, two cases are discussed in detail. Of the 10 US Air Forces persons in central peninsular Malaysia (Arness et al., 1999), a 35 year old previously healthy individual reported mild diarrhea, muscle and joint pain, fleeting skin rashes, night sweats, chills, fever, weight loss, and muscle wasting (hollow indentations on skull), but no lymphadenopathy, often a hallmark of acquired toxoplasmosis. Four months later, dermal nodules were observed on his elbows, abdomen, and limbs (Arness et al., 1999). A few sarcocysts were seen in both muscle biopsies, often without inflammation (Fig. 5). The presence of metrocytes in sarcocysts indicated recently acquired infection but the species of Sarcocystis responsible could not be determined.

Another example is of a 32 year old woman from China who was among 92 college students and teachers on vacation on Pangkor Island (Italiano et al., 2014). She had headache, myalgia, arthralgia, and 3 rounds of relapsing fever. The most prominent symptom was facial swelling. Magnetic resonance imaging of her face revealed asymmetrical appearance of the temporalis

Fig. 4. TEM of sarcocyst walls of five Sarcocystis species in cattle. Note br = bradyzoite, hc = host cell, gs = ground substance layer, se = septum, vp = villar protrusions. (A) S. cruzi. The vp are long, lack microtubules, and folded over the sarcocyst wall. (B) S. heydorni. The vp are triangular and have disc-like structures (arrowheads). (C) S. hirsuta. Note expanded villar protrusions with a narrow stalk (arrows) and thick ground substance layer. (D) S. hominis. The villar protrusions are upright, finger-like, and the gs is thick. (E) S. rommeli. The vp are elongated, irregular in thickness, have vesicles at the base, and the gs is thin.

Table 3

Sarcocystosis in travelers returned from Malaysia.

No. of Dates of travel Location In Malaysia Initial symptoms Muscle biopsy Reference

persons

7 of 10a May, 1993 Village, 80 km Northeast of Kuala Lumpur Acute fever, bronchospasm, myositis, muscle wasting, arthralgias, fatigue, eosinophilia Sarcocysts in 1 patient biopsied Arness et al. (1999)

68 of October, 2011 to Tioman Island Myalgia, fatigue, fever, headache, Sarcocysts in 6 of 15 patients biopsied. Esposito

99b April, 2013 arthralgia, nausea, diarrhea, cough, rash, night sweats, chest pain, swelling, pruritis, chills in decreasing order PCR-positive Sarcocystis in 1 biopsy et al. (2014),e

89 of 17 to 19 January, Pangkor Island Fever, myalgia, headache, cough, joint Sarcocysts in histologic sections of3 Italiano et al.

92c 2012 pain, nausea, vomiting, diarrhea, rash in decreasing frequency. Facial swelling is prominent in some patients. muscle biopsy. PCR positive on 1 muscle biopsy not examined histologically and 2 histologically confirmed cases. (2014),f

6d March-April, 2014 Tioman Island Fever, headache, myalgia No muscle biopsy Tappe et al. (2014), Tappe et al. (2015)

a Adult males U.S. Air Force Special Tactics team.

b Patients were from Germany 29, France 23, The Netherlands 6, Switzerland 3, Belgium 2, Spain 2, Italy 1, Canada 1 and Singapore 1.

c College students and teachers on a retreat in a hotel; 73 Malaysians, 21 non- Malaysians (9 from China, 3 from Nepal, 2 Korea, 2 Indonesia, 1 India, 1 Netherlands, 1 Myanmar, 1 Iran). d Germany.

e Other papers discussing this group of patients: CDC (2012), Esposito et al. (2012). Husna Maizura et al. (2012), Tappe et al. (2013a,b), Tappe et al. (2015), Harris et al. (2015), Slesak et al. (2015), Esposito et al. (2015). f Other papers discussing this group of patients: AbuBakar et al. (2013), Lau et al. (2014), Italiano et al. (2015).

muscle. She had blood eosinophilia, and elevated liver and muscle enzymes. Muscle biopsy revealed S. nesbitti-like sarcocyst by histological and PCR testing (Italiano et al., 2014).

22.2.1. Evidence to possibly link S. nesbitti with human sarcocystosis or lack of it

S. nesbitti was named by Mandour (1969) for sarcocysts found in 2 of 44 Macaca mulatta (Rhesus monkey) from India. Sarcocysts were found in skeletal muscles, but not in the heart. They were up to 1100 |jm long. The light microscopic description is vague, an ultrastructural examination of the sarcocysts was not made. Attempts to locate original specimens, reported to be deposited in the London School of Hygiene and Tropical Medicine, were unsuccessful (J. P. Dubey, own investigations, 2014).

A S. nesbitti-like parasite was found in 1 of 69 Macaca fascicularis that were reported to be born on a farm in China (Yang et al., 2005). Macaca fascicularis is native to India, Philippines, Malaysia, Indonesia, Vietnam, Cambodia, Laos, Thailand, and Myanmar, and it is uncertain if these monkeys were imported to China (Yang — personal communication to JPD August, 2015). S. nesbitti-like sarcocysts were 2.3 to 14 mm long, and up to 0.1 mm wide (Fig. 5). By light microscopy (LM) the sarcocyst wall was thin and smooth. By transmission electron microscopy (TEM) the sarcocyst wall had small vesicular invaginations, 19-380 nm in diameter and 65-120 nm in depth, type 1 (Dubey et al., 2015a). Bradyzoites were 7.5-10 x 1.25 |jm by LM, and 3.3-6.5 x 1.0-2.79 |jm by TEM. The characterization of 18S rRNA gene from DNA extracted from 2 unfixed S. nesbitti sarcocysts from this monkey from China by Tian et al. (2012) indicated a close relationship to S. singaporensis, which cycles between pythons and rodents, suggesting that snakes are likely a definitive host for S. nesbitti. The genetic sequences from S. nesbitti from the monkey matched the sequences derived from a sarcocyst from three symptomatic cases of Sarcocystis in humans from Pangkor Island, Malaysia (Lau et al., 2014, Table 3). The DNA sequences for Sarcocystis from these patients varied from each other by 1% and a BLAST result found that they shared 99% homology with S. nesbitti from the muscle of M. fascicularis. However, data based solely on 18S rRNA gene are of limited taxonomic value (Dubey et al., 2015a).

It is probable that snakes may act as definitive hosts for S. nesbitti, because, recently, sequences amplified from the 18S rDNA genes were found matching this species in sporocysts excreted by reticulated python (Braghammerus reticulatus syn. Python reticulatus) and monocled cobra (Naja kaouthia) from Malaysia (Lau et al., 2013, 2014). A presumptive cycle is depicted in Fig. 6.

Linking the mysterious disease in humans noted in the previous section to sarcocystosis faces many challenges. The following lessons learnt from the review of biology of Sarcocystis species in animals (Dubey et al., 2015a) might be helpful in future investigations.

1. There are more than 200 species of Sarcocystis, with many variations in biology and life cycles. Most species are host-specific with limited host range while some (e.g. S. neurona) have wide host range. Therefore, caution should be used to extrapolate information from one Sarcocystis species to other Sarcocystis species.

2. Clinical sarcocystosis in animals is associated with schizogonic phase, and by the time sarcocysts mature, animals often become asymptomatic. The sarcocyst burden in muscle is not a reliable criterion to gauge clinical disease. Some animals remain asymptomatic even when 25% of muscle mass is due to sarcocysts (JPD unpublished observations). The schizogonic phase has not been found in any report of Sarcocystis infection in humans. Although schizonts in most Sarcocystis species are time-limited (lasting 1-4 weeks), schizonts of some species may persist for several months (e.g. S. falcatula of birds). Parasitemia occurs

Fig. 5. Sarcocystis nesbitti-like sarcocysts in skeletal muscle biopsies from three humans with clinical sarcocystosis. (A, B, C) light microscopy, hematoxylin and eosin stain. The sarcocysts are thin walled (arrows). (D) TEM. The parasitophorous vacuolar membrane has tiny blebs (opposing arrowheads), and gs is relatively thin. (For sources of figures see Dubey et al., 2015a).

following schizont rupture, and merozoites can be detected in peripheral blood. The number of merozoites in blood is few, therefore examination of buffy coats for merozoites during the febrile stage in humans may facilitate the detection of DNA and/or intact merozoites of the S. nesbitti-like parasite in the early stage of infection. Peripheral eosinophilia observed in human cases (Table 3) is not a feature of sarcocystosis in animals.

3. Most, but not all species of Sarcocystis cross-react serologically or immunohistochemically. Therefore, undue reliance on these methods should be avoided until the full life cycle of S. nesbitti is definitively known. I have received many requests from patients and their clinicians who were ill of undiagnosed disease but wanted to be tested for Sarcocystis neurona because they were misinformed or their horse had been diagnosed with S. neurona infection (J.P. Dubey, unpublished observations). Part of this might be related to the detection of S. neurona antigen in human serum using Western blott (see Arness et al., 1999) which may be unreliable.

4. Biologically and structurally distinct Sarcocystis species can be very closely related phylogenetically (examples S. neurona, S. falcatula). Therefore, full molecular characterization of S. nesbitti-like parasite in humans and from the implicated primate intermediate-host species is awaited before the two parasites can be linked.

5. Some carnivores are definitive hosts for numerous species of Sarcocystis, but species transmitted by canids are usually not transmitted by cats. Therefore, it is unlikely that S. nesbitti will have limited definitive hosts.

Fig. 6. Presumed life cycle of Sarcocystis nesbitti-like parasite with humans as aberrant host. (From Fayer et al., Clin. Microbiol. Rev. 28:295-311, 2015).

6. Sarcocystis species with livestock as intermediate hosts have bradyzoites about twice the size of bradyzoites in Sarcocystis species of small mammals, and poikilothermic animals. The bradyzoites of the S. nesbitti-like parasite in humans are small in size, suggesting poikilothermic animals as definitive host.

7. Sarcocyst is the dead-end (or accidental) phase of the life cycle of Sarcocystis in the intermediate host. Although sarcocysts rupture or degenerate periodically, released bradyzoites are not known to multiply or initiate new infection in any host. Clinical illness in humans is more likely associated with schizonts or immature sarcocysts, and immune responses may aggravate clinical disease (Olias et al., 2013).

There is an urgent need to study life cycles of Sarcocystis species in primates, particularly, those infecting macaques in their natural habitat, and full molecular characterization of S. nesbitti-like parasite. The identity of S. nesbitti in humans is uncertain. We recently summarized available literature on Sarcocystis infections in human primates to facilitate further studies (Dubey et al., 2015a).

Conflict of interest

Acknowledgments

1 thank Drs. Douglas Esposito, Dennis Tappe, Claire Italiano, Gaston Moré, Rafael Calero-Bernal, Camila Cézar, and Shiv Kumar Verma for their help in preparation of this paper.

The views expressed are of the author and not the United States Department of Agriculture.

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