Ancient Human Parasites in Ethnic Chinese Populations

Whilst archaeological evidence for many aspects of life in ancient China is well studied, there has been much less interest in ancient infectious diseases, such as intestinal parasites in past Chinese populations. Here, we bring together evidence from mummies, ancient latrines, and pelvic soil from burials, dating from the Neolithic Period to the Qing Dynasty, in order to better understand the health of the past inhabitants of China and the diseases endemic in the region. Seven species of intestinal parasite have been identified, namely roundworm, whipworm, Chinese liver fluke, oriental schistosome, pinworm, Taenia sp. tapeworm, and the intestinal fluke Fasciolopsis buski. It was found that in the past, roundworm, whipworm, and Chinese liver fluke appear to have been much more common than the other species. While roundworm and whipworm remained common into the late 20th century, Chinese liver fluke seems to have undergone a marked decline in its prevalence over time. The iconic transport route known as the Silk Road has been shown to have acted as a vector for the transmission of ancient diseases, highlighted by the discovery of Chinese liver fluke in a 2,000 year-old relay station in northwest China, 1,500 km outside its endemic range.     

A review of Gymnophalloides seoi (Digenea: Gymnophallidae) and human infections in the Republic of Korea.

Studies on Gymnophalloides seoi (Digenea: Gymnophallidae) and human infections are briefly reviewed. This minute intestinal fluke was first discovered from a Korean woman suffering from acute pancreatitis and gastrointestinal troubles. It was described as a new species by Lee, Chai and Hong in 1993. The southwestern coastal village where the patient resided was found to be a highly endemic area, and additional endemic areas have been identified. The parasite is very small, 0.33-0.50 mm long and 0.23-0.33 mm wide, and characterized by the presence of a ventral pit. The first intermediate host remains unknown, but the second intermediate host has been found to be the oyster Crassostrea gigas. Man and the Palearctic oystercatcher Haematopus ostralegus have been shown to be natural definitive hosts, and wading birds including the Kentish plover Charadrius alexandrinus are highly susceptible to experimental infection. Gerbils, hamsters, cats, and several strains of mice were also susceptible laboratory hosts. In experimentally infected mice, the parasites inhabit the small intestine, pinching and sucking the root of villi with their large oral suckers, but they did not invade beyond the mucosa in immunocompetent mice. However, they were found to invade the submucosa in immunosuppressed mice. Human G. seoi infections have been found in at least 25 localities; 23 islands on the Yellow Sea or the South Sea, and 2 western coastal villages. The highest prevalence was found in a village on Aphaedo, Shinan-gun (49% egg positive rate); other areas showed 0.8-25.3% prevalence. Infected people complained of variable degrees of gastrointestinal troubles and indigestion. The infection can be diagnosed by recovery of eggs in the feces; however, an expert is needed to identify the eggs. Praziquantel, 10 mg/kg in single dose, is effective for treatment of human infections. Eating raw oysters in endemic areas should be avoided.     

Neotropical schistosomiasis: African affinities of the host snail Biomphalaria glabrata (Gastropoda: Planorbidae)

Schistosoma mansoni , the blood fluke responsible for human intestinal schistosomiasis in the Neotropics, was imported repeatedly with African slaves during the period 1500–1800. This trematode, and its intermediate host snails of the genus Biomphalaria , are widely distributed across Africa and the disease is thought to have quickly become established in South America and the West Indies because of the presence of an endemic susceptible congener, B. glabrata. We compared B. glabrata with four other Neotropical and three African species of Biomphalaria using 20 allozyme loci and found that it is phenetically and phylogenetically more like the African species; both parasite and American host snail are apparently of historically or geologically recent African origin. Furthermore, genetic distances, cladistic analyses and fossil data suggest the African Biomphalaria species may themselves have evolved from Neotropical founders following an initial trans-Atlantic dispersal in the reverse direction 2.3–4.5 Mya. Interpretation of existing patterns remains problematic as few African snails have been characterized genetically and both B. glabrata and African B. pfeifferi appear to comprise several cryptic species.     

High prevalence of Fasciolopsis buski in an endemic area of liver fluke infection in Thailand

There is a high prevalence of Fasciolopsis buski in Sawasdee Village in the Nam Som District, Udonthani Province in northeastern Thailand, an endemic area of liver fluke infection. From stool examination of 183 villagers participating in the study, the authors detected a fluke parasitic infection rate of 14.8% (27 cases). Of interest, the authors found a similar high prevalence of both Opisthorchis viverrini (16 cases, 8.7%) and F buski (13 cases, 7.1%). This region is the endemic area of liver fluke, not of F buski. The surrounding environment is the hill area of Nayoong Namsom mountain, which is not a suitable area for F buski larvae. However, the village has been inhabited by a number of immigrants from various regions of Thailand over the past few years. The F buski can be carried by these people, possibly explaining the migration of the fluke parasite to this setting. The nonendemic parasite infection was also detected at high prevalence in the community. Wide spectrum control for various parasitic infections in villages like Sawasdee is necessary.     

Clonorchis sinensis and clonorchiasis, an update

Clonorchis sinensis is the most common human liver fluke in East Asia. Several studies proved its carcinogenesis in humans and it was reclassified as a group 1 biological carcinogen in 2009. It is still actively transmitted in endemic areas of Korea, China, Russia, and Vietnam. Currently it is estimated that more than 200 million people are at risk of infection, 15-20 million people are infected and 1.5-2 million show symptoms or complications. Several molecules and genes of the fluke have been identified and characterized. Studies on its oncogenesis and omics-based findings have been especially encouraging. Diagnosis of its infection depends mainly on detection of eggs in feces but other methods have been developed. ELISA using crude extract antigen is now popular for its diagnosis. Diagnosis by detecting DNAs from eggs in feces has been developed using PCR, real-time PCR, and LAMP, which have been found sensitive and specific. Imaging diagnosis has been studied in depth and is widely used. Any evidence of clonorchiasis, such as eggs, DNAs, or images, may lead to recommendations of chemotherapy in endemic areas. Praziquantel is the major chemotherapeutic agent for clonorchiasis and recently tribendimidine was found effective and is now under investigation as a promising chemotherapeutic alternative. Sustainable control programs which include mass chemotherapy with praziquantel and education for prevention of re-infection may reduce its morbidity and eliminate its infections in endemic areas.     

A new species of Tricula (Pomatiopsidae: Triculinae) from Sichuan Province,PR China: Intermediate host of Schistosoma sinensium

A new species of pomatiopsid gastropod, Tricula hortensis, is described from Sichuan Province, PR China. This new species is characterized by a large columellar peg or tooth situated mid body‐whorl. The reproductive anatomy of the female shows a number of character states that are plesiomorphic for the Triculinae. Tricula hortensis may be distinguished from the two other species of Tricula which bear a columellar tooth by differences in the shell and in pallial and/or reproductive structures. Tricula hortensis is found in spring‐fed irrigation canals serving market gardens in Mianzhu County, Chengdu, Sichuan. This species acts as intermediate host for the parasitic blood fluke Schistosoma sinensium Bao 1958.     

Aroapyrgus alleei Morrison, 1946 (Gastropoda: Hydrobiidae) first intermediate host of paragonimus mexicanus in Colima, Mexico

The lung fluke Paragonimus mexicanus infects opossums in the state of Colima, on the Pacific coast of Mexico. Some of the hydrobiid snails occurring in the same area, and identified as Aroapyrgus alleei Morrison, 1946, were found naturally infected with rediae and cercariae of P. mexicanus. The morphology of A. alleei is here described and compared to that of other species of Aroapyrgus, which are actual or potential hosts of lung flukes in other enzootic or endemic areas in Central and South America.     

When endemic coral‐reef fish species serve as models: endemic mimicry patterns in the Marquesas Islands

This article documents several cases of widespread species, which usually mimic other widespread species throughout the Indo‐Pacific, using endemic Marquesan species as a model and displaying endemic mimicry patterns. This discovery adds a new line of evidence to the uniqueness of the Marquesas Islands, which not only host a high number of endemic reef‐fish species, but also endemic mimicry patterns.     

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis

Background

Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.

Results

We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.

Conclusions

This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.     

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.     

Fas2-ELISA in the detection of human infection by Fasciola hepatica

Fasciola hepatica has recently emerged as a major pathogen of humans from reports on areas of endemicity and hyper-endemicity for fascioliasis. This situation is aggravated by the lack of standard assays for the screen diagnosis of F. hepatica infection in humans living in endemic areas. Our laboratory has developed an enzyme-linked immunosorbent assay (Fas2-ELISA) based on the capture of IgG antibody by a purified protein Fas2, which is an adult fluke cysteine proteinase. Fas2-ELISA exhibited 95% sensitivity and 100% specificity in 38 individuals infected with F. hepatica diagnosed by finding eggs in stools and 46 serum samples from healthy volunteers. No cross-reaction was observed with 54 serum samples from patients with ten different parasitic infections including the trematodes Paragonimus westermani and Schistosoma mansoni. The high antigenicity of Fas2 is suggested by the fact that antibodies to Fas2 rise rapidly by 1-2 weeks of infection and rise until patency at 8 weeks of infection in experimentally infected alpacas. Field screening for human fascioliasis using Fas2-ELISA and coprology in three endemic locations of the Peruvian Andes resulted in 95.5% sensitivity, 86.6% specificity in a population of 664 children in an age range of 1 to 16 years old. These results provide evidence of the clinical potential of Fas2-ELISA to diagnose fascioliasis in humans exposed to liver fluke infection in endemic areas for this parasite. Fas2-ELISA is currently developed as a standard assay for both field screening for fascioliasis in people living in endemic areas and detecting occasionally F. hepatica infected patients in clinical laboratories.     

Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.     

Seasonal dynamics of Fasciola hepatica burdens in grazing Timahdit sheep in Morocco

Seasonal transmission of Fasciola hepatica was observed in sentinel sheep and the dynamics of the snail intermediate host, Lymnaea truncatula, was followed over a 3-year study period in the Middle-Atlas mountains in Morocco. High fluke burdens were recorded in both lambs and ewes in the fall and winter, suggesting that transmission occurred in late spring. Fluke burdens ranged from one to 302 in ewes and from one to 345 in lambs. Infections with 200 or more flukes were always fatal. A unique feature of this study was the annual cyclical fluctuation of the fluke burdens. Burdens reached maximum levels during the winter and then declined to low numbers by late spring and summer. This suggested self-regulation which may be dependent on breed resistance or may be related to forage factors, including lack of forage (nutritional stress). Snail populations were cyclical and correlated with fluke transmission as observed in the sentinel sheep. The weather was observed to affect the snail populations which in turn limited fluke transmission.     

Composition and metabolism of phospholipids of Fasciola hepatics, the common liver fluke.

1. The phospholipid composition of Fasciola hepatica, the common liver fluke, was compared to that of the liver of the host animals (rats and cattle). Considerable differences were found:monoacyl-sn-glycero-3-phosphorylcholine, hardly detectable in the liver, was found in significant amounts in the parasite. On the other hand, sphingomyelin, a normal constituent in the liver, appears to be absent in the liver fluke. Fasciola hepatica isolated from rat and cow liver had a strikingly similar phospholipid composition. 2. Qualitative and quantitative differences were also found between the fatty acyl constituents of the phospholipids of the parasite and the liver. The major difference was the presence of eicosaenoic and eicosadienoic acids in the parasite, whereas these acids were not detected in the liver. 3. In vitro incubations of Fasciola hepatica in the presence of (32P)phosphate and (2-3H)glycerol resulted in the labelling of all phospholipids of the parasite, except that the 3H label did not incorporate into ethanolamine plasmalogen. This is in agreement with the concept that in animals, glycerol is introduced into plasmalogens via dihydroxyacetonephosphate. 4. Homogenates of liver flukes were found to catalyze the synthesis of phosphatidylcholine from 1,2-diacyl-sn-glycerols and CDPcholine. 5. These results strongly suggest that Fasciola hepatica is capable of synthesizing at least part of its fatty acids and phospholipids.     

Patterns of coccidial prevalence in lizards of Mauritius

This paper reports prevalence of coccidial oocysts in fecal samples from 6 endemic and 2 introduced lizard species on Mauritius, an island nation in the Indian Ocean. Total coccidian prevalence was 54% (n = 341) for the endemic 'ornate day gecko,' Phelsuma ornata; 48% (n = 159) for the endemic 'Durrell's night gecko,' Nactus durrelli; 53% (n = 15) for the endemic 'Serpent Island night gecko,' N. serpensinsula; and 78% (n = 248) for the introduced gecko, Hemidactylus frenatus. These high prevalences may reflect lack of long-lasting immune response to coccidial infection. There were few significant differences in prevalence among age, island, sex, or body condition within species, suggesting that these coccidia are relatively nonpathogenic and have little effect on host fitness. Prevalence was higher in the dry season than the wet season. These data suggest other factors, such as low host immune response to reinfection, affect overall prevalence more significantly than the effect of humidity on oocyst survival on Mauritius. No coccidia were found in samples from the endemic 'Gunner's Quoin night gecko,' N. coindemirensis (n = 155), probably reflecting parasite extinction due to a host population bottleneck following historical introduction of rats. There was no evidence of competitive or facilitative interactions between Eimeria sp. and Isospora sp., but evidence of competition between 2 Eimeria species in the 'ornate day gecko,' Phelsuma ornata. No evidence was found of cross-species infection, suggesting that reptile coccidia have high host specificity and are, therefore, poor subjects for studies of parasite-mediated competition and the evolution of sex.     

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.     

Tolerance of liver fluke infection varies between breeds and producers in beef cattle

Liver flukes (Fasciola spp.) are important helminth parasites of livestock globally and cause substantial reductions in health and productivity of beef cattle. Attempts to control fluke have been thwarted by the difficulty of vaccine design, the evolution of flukicide resistance and the need to control the intermediate snail host. Mechanisms to reduce the impact of parasites on animal performance have typically focused on promoting host resistance – defined as the ability of the host to kill and remove the parasite from its system – and such strategies include improving protein nutrition or selective breeding for resistance. Organisms, however, have another broad mechanism for mitigating the impact of parasites: they can show tolerance, defined as the ability to maintain health or performance under increasing parasite burden. Tolerance has been studied in the plant literature for over a century, but there are very few empirical studies of parasite tolerance in livestock. In this study, we used data collected from > 90 000 beef cattle to estimate the impact of the severity of liver fluke infection on performance and variation in tolerance of fluke. Severity of liver fluke infection was estimated using liver “fibrosis score” on a scale of 0–3 and performance estimated as (1) age at slaughter and (2) daily dead weight gain. Animals with higher fibrosis scores were slaughtered around 2 weeks later than animals with no fluke and gained around 10 g less weight per day. There was also considerable variation in these effects of fibrosis score, such that animals from different producers and breeds varied in their tolerance of fluke infection. While breeds did not vary in the association between fibrosis and age at slaughter, there was considerable variation among producers: high fibrosis score delayed slaughter by up to 50 days in some producers, but not at all in others. Meanwhile, there was support for variation in the slope of daily dead weight gain on fibrosis score among both breeds and producers, with some unaffected by high fluke scores and some breeds and producers experiencing a 20 g/day lower weight gain under high fluke scores. Our results point to the potential for both environmental and genetic variation in tolerance of liver fluke in cattle, paving the way for quantitative genetic and nutritional research into the feasibility of promoting tolerance as a disease mitigation strategy.     

ROBERTSIELLA SILVICOLA, A NEW SPECIES OF TRICULINE SNAIL (CAENOGASTROPODA: POMATIOPSIDAE) FROM PENINSULAR MALAYSIA, INTERMEDIATE HOST OF SCHISTOSOMA MALAYENSIS (TREMATODA: DIGENEA)

A new species of pomatiopsid gastropod, Robertsiella silvicola,is described from Perak, peninsular Malaysia. The encapsulationof the ducts of the bursa copulatrix complex, short (wide) spermathecalduct and penial stylet indicate that this is a species of Robertsiella.Robertsiella silvicola is distinguished from other Robertsiellaby its small size, the lack of pronounced spiral macrosculptureon the shell, and the nature of the stylet. Robertsiella silvicolais found in water bodies ranging from small, spring-fed, first-orderstreams to tiny trickles of water draining off forested hillsides.This species appears restricted to limestone areas in the foothillsof the mountain chains of Kedah and Perak States, West Malaysia.It appears to be a highly compatible intermediate host for theparasitic blood fluke Schistosoma malayensis Greer, Ow-Yang& Yong, 1980 that infects humans and other mammals in Malaysia. (Received 15 September 2004; accepted 21 March 2005)     

Advances in Fasciola hepatica research using ‘omics’ technologies

The liver fluke Fasciola hepatica is an economically important pathogen of livestock worldwide, as well as being an important neglected zoonosis. Parasite control is reliant on the use of drugs, particularly triclabendazole, which is effective against multiple parasite stages. However, the spread of parasites resistant to triclabendazole has intensified the pursuit for novel control strategies. Emerging 'omics' technologies are helping advance our understanding of liver fluke biology, specifically the molecules that act at the host-parasite interface and are central to infection, virulence and long-term survival within the definitive host. This review discusses the technological sequencing advances that have facilitated the unbiased analysis of liver fluke biology, resulting in an extensive range of ‘omics’ datasets. In addition, we highlight the ‘omics’ studies of host responses to F. hepatica infection that, when combined with the parasite datasets, provide the opportunity for integrated analyses of host-parasite interactions. These extensive datasets will form the foundation for future in-depth analysis of F. hepatica biology and development, and the search for new drug or vaccine interventions.