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PCR diagnosis of infections with different species of Opisthorchiidae using a rapid clean-up procedure for stool samples and specific primers.

Infections with the opisthorchiid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and Opisthorchis felineus cause serious health problems in endemic areas of Southeast Asia and countries of the former Soviet Union. Chronic infections--even with low worm burdens--may lead to the development of fatal cholangiocarcinoma and related symptoms. A more sensitive diagnosis is needed since the tiny eggs of the worms are often not seen in microscopic examinations of stool samples, especially in patients with low infections. This communication reports a rapid cleanup procedure for human stool samples, which enables reliable identification of DNA by polymerase chain reaction from few eggs of opisthorchiid flukes in fecal samples.

Animals↗

Detection of circulating Fasciola gigantica antigen in experimental and natural infections of sheep with fasciolosis.

Detection of circulating Fasciola gigantica antigen was performed in sera of sheep with experimental and natural F. gigantica infections using the direct enzyme-linked immunosorbent assay method. Sera from sheep with monoinfections of Schistosoma bovis, Dicrocoelium hospes and Paramphistomum microbothrium were included in the assay to ascertain specificity. Circulating F. gigantica antigen (CFA) was detected as early as 1 week after infection in the experimentally infected sheep. No detectable CFA was observed 2 weeks after chemotherapy. Positivity rates of 82.5%, 12.5%, 10% and 10% were found in sera with monospecific infections of F. gigantica, P. microbrothrium, D. hospes and S. bovis, respectively. Acid treatment of the sera did not enhance the sensitivity of the assay.

Animals↗

Life cycle and morphology of Metagonimus miyatai (Digenea: Heterophyidae) from Nagano, Japan.

The life cycle of Metagonimus miyatai Saito, Chai, Kim, Lee and Rim, 1997 (Digenea: Heterophyidae) was studied in the Hiroi River at Kotobuki, Iiyama City, Nagano Prefecture, central Japan. Daughter rediae and cercariae were found in Semisulcospira libertina (Gould) and S. dolorosa (Gould) (Gastropoda: Pleuroceridae). Metacercariae were found encysted in Phoxinus lagowskii steindachneri Sauvage (Osteichthyes: Cyprinidae). Cercariae were experimentally exposed to P. lagowskii steindachneri, and metacercariae were obtained from the fish. Metacercariae of natural and experimental infections were experimentally fed to respective golden hamsters, and gravid adults identifiable as M. miyatai were recovered from the small intestine of the golden hamsters. The daughter redia, cercaria, metacercariae and adults are described. The life cycle of M. miyatai is discussed.

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Diplostomum spathaceum (Rud. 1819): effects of physical factors on the infection of rainbow trout (Salmo gairdneri) by cercariae.

The relationship between infection rates of rainbow trout (Salmo gairdneri) by Diplostomum spathaceum and cercarial concentration, water flow rate and temperature were investigated by means of controlled infections within a flume. A linear relationship was obtained between cercarial concentration and mean abundance of metacercariae/fish. A biphasic relationship occurred between flow rate and abundance of metacercariae. Within the confines of the flume, it was possible to control the infection rate of trout with D. spathaceum cercariae by manipulating flow rate, suggesting that it may be a possible method of controlling diplostomiasis on fish farms. No infection occurred in fish infected and maintained below 10 degrees C and the optimum infection temperature was approximately 17.5 degrees C. Infections became established in fish infected at 7.5 and 5 degrees C but maintained at 15 degrees C prior to examination. Trout were infected at 7.5 degrees C for 10-50 min and all attached cercariae were washed off and removed from the flume. Following infection, fish were either maintained at 7.5 degrees C or 15 degrees C prior to examination. Using this method, it was possible to ascertain that it was migration and not penetration which was inhibited at water temperatures below 10 degrees C.

Animals↗

Prevalence of Opisthorchis viverrini infection among villagers harboring Opisthorchis-like eggs.

The precise occurrence of Opisthochis viverrini infection in humans, who were positive for Opisthorchis-like eggs in a stool examination, was determined using the potassium permanganate staining method. In the 68 specimens examined, there were more individual O. viverrini eggs (38.24%) than singular Haplorchis taichui eggs (29.41%). One-fourth of the total specimens contained a mixed infection of O. viverrini and H. taichi eggs. The median ratio of O. viverrini: H. taichui eggs in mixed infection cases was 2.29 (min = 1, max = 17.5). It is suggested that chemotherapy with praziquentel treatment should be given to patients who are positive for O. viverrini-like eggs.

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Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle.

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.

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Maternal transmission by Alaria marcianae (Trematoda) and the concept of amphiparatenesis.

The term amphiparatenic host was coined originally for hosts of Alaria marcianae that as adults are paratenic hosts, but as juveniles serve as definitive hosts. In this study the concept of amphiparatenesis is placed in a theoretical context, maternal transmission shown to be the basic mechanism, and the concept extended to include Toxocara canis, T. cati, T. pteropodis, Neoascaris vitulorum, Ancylostoma caninum, Uncinaria lucasi, various species of Strongyloides, Pharyngostomoides procyonis, and P. adenocephala. Alaria marcianae mesocercariae were used in a feline model to show that male and nonlactating female cats are definitive hosts, but lactating cats are primarily paratenic hosts. Inoculation of 1 female cat resulted in the infection of 21 of her offspring via the milk over the course of 5 litters and after a 3-yr period she still had viable larvae in her tissues. The ability of parasites to remain immature in amphiparatenic hosts is believed to be an adaptation on the part of the parasite to promote dissemination through maternal transmission and not the result of resistance, immunological or otherwise, on the part of the host. The amphiparatenic concept has important implications that include: the use of pregnant and lactating females as reservoirs of infection for the offspring; infection transmitted through a contagious transplacental or transmammary pathway; a parasite population structure in which adult worms are in greater abundance in neonate than adult hosts; and the effective control of parasites utilizing this strategy proving to be very difficult.

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Susceptibility of various species of animals and strains of mice to Gymnophalloides seoi infection and the effects of immunosuppression in C3H/HeN mice.

Susceptibility to Gymnophalloides seoi infection was studied in 8 species of animals, including 7 strains of mice; the effects of immunosuppression on susceptibility were examined in C3H/HeN mice. One hundred metacercariae of G. seoi isolated from naturally infected oysters were orally administered to each animal. Worm recovery rate (WRR), worm dimensions, and the number of uterine eggs were obtained at day 3 and day 7 postinfection (PI). Average WRR from gerbils, hamsters, and cats at day 7 PI was 28.0%, 14.2%, and 10.9%, respectively, the former 2 figures of which were significantly higher than the rate of 0.0-4.0% from Sprague-Dawley rats, dogs, ducks, guinea pigs, and chicks. In the case of mice, average WRR at day 7 PI was 12.4% (KK strain), 11.8% (C3H/HeN), 9.6% (ICR), 6.4% (BALB/c), and 6.3% (ddY), respectively; the first 3 figures were significantly higher than the rates from other strains, which were 1.8% (A) and 0% (C57BL/6). At day 3 PI, WRR was much higher in all strains except C57BL/6. Worm maturation was the highest in C3H/HeN mice. Immunosuppression of C3H/HeN mice by injecting prednisolone for 7, 14, or 21 days prior to infection increased WRR at day 7 PI to 27.8%, 33.8%, or 67.5%, respectively. The results show that gerbils, hamsters, cats, and KK, C3H/HeN, ICR. BALB/c, and ddY mice are laboratory hosts that are fairly susceptible to G. seoi infection. In C3H/HeN mice, susceptibility was markedly enhanced by immunosuppression.

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Effects of Trichobilharzia ocellata on hemocytes of Lymnaea stagnalis.

We analyzed the effects of infection with Trichobilharzia ocellata on hemocytes of its snail host, Lymnaea stagnalis, and correlated them with successive stages of parasite development. Circulating hemocytes were studied at 0, 2, 4, 6, and 8 weeks post exposure (p.e.) with respect to cell number, distribution of subpopulations (as characterized by morphology, determinants recognized by either of two lectins and a monoclonal antibody) and to proliferative, phagocytic and endogenous peroxidase activity. Infection results in a net elevated level of activity of circulating hemocytes at 2 weeks p.e., when mother sporocysts are present in the head-foot-mantle region, as well as at 4 weeks p.e., when daughter sporocysts are migrating to and growing in the digestive gland region. A lower level of activity was observed at 6 weeks p.e., when cercariae are differentiating within daughter sporocysts. A net activation was again found at 8 weeks p.e., when cercariae are escaping. So, infection with T. ocellata results in a net general activation of the internal defense system of L. stagnalis, during several stages of development of the parasite.

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Proterometra macrostoma (Digenea: Azygiidae): variations in cercarial morphology and physiology.

Snails, Elimia semicarinata, infected with Proterometra macrostoma were collected monthly in 1990 and 1991 from North Elkhorn Creek near Lexington, Kentucky, and kept on a 12:12 h light-dark cycle for 2 weeks. Cercariae emerging from snails were classified into 8 strains (I-VIII) based on differences in number and distribution of spined and spineless papillae on the tail. Cercariae also had unique patterns of emergence, swimming behaviour and infectivity in 4 species of sunfish. Of 513 infected snails collected in May, 339 had pure infections with the strain frequencies (% of 339): I, 46.6; II, 7.7; III, 12.1; IV, 8.8; V, 0.6; VI, 2.7; VII, 11.8; VIII, 9.7. In the multiple infections, 159 snails shed 2 strains, 14 shed 3, and 1 snail shed 4 strains simultaneously. A comparison of sunfish and parasite populations in Kentucky, Ohio and Michigan indicated that strain frequency in P. macrostoma is regulated by the species composition of the sunfish population.

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Morphology and life history of Petasiger variospinosus (trematoda: echinostomatidae) in the Free State, South Africa.

Specimens of the freshwater snail Bulinus tropicus (Krauss, 1848) collected in the Free State, South Africa shed cercariae with an oral collar bearing 27 spines. Tadpoles of the African clawed toad Xenopus laevis laevis Daudin, 1802 collected from the same waters harbored metacercariae with a similar collar of spines. Adults were obtained after feeding infected tadpoles to laboratory-reared reed cormorants, Phalacrocorax africanus (Gmelin, 1789). The parasite was identified as Petasiger variospinosus (Odhner, 1910), the life cycle was experimentally completed, and stages described by the use of light and scanning electron microscopy.

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The palearctic oystercatcher Haematopus ostralegus, a natural definitive host for Gymnophalloides seoi.

In order to verify the role of migrating birds as natural definitive hosts for Gymnophalloides seoi (Gymnophallidae), Palearctic oystercatchers, Haematopus ostralegus, were caught from several western coastal areas in the Republic of Korea and examined for intestinal flukes. Five (71.4%) of 7 oystercatchers were infected with 302-1,660 (mean 892) adult G. seoi. In intestinal sections of the host, worms were found in the intervillous space of the mucosal layer. We conclude that the oystercatcher is a natural definitive host for G. seoi.

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[Intestinal flukes of genus Metagonimus and their second intermediate hosts in Kangwon-do].

This study investigate the epidemiological feature of Metagonimus infection in Kangwon-do (province). The average infection rate of the surveyed inhabitants was 7.8% (83 positives out of 1, 067 examinees) by stool examination; male, 11.4% and female, 3.2%, respectively. The egg positive rate in residents in the Som river area was 7.3%, that of the Chuchon river area 6.3%, the Pyongchang river area 12.8%, the Tong river area 3.8%, the Hongchon river area 9.8%, and the Ohsip stream area of Samchok 8.0%, respectively. The average metacercarial infection rate of genus Metagonimus in the fish was 81.0% (256 positives out of 318 fish). The infected fishes were Zacco platypus, Zacco temmincki, Opsariichthys bidens, Squalidis sp., Carassius carassius, etc. in western Kangwon-do Meanwhile, in the Ohsip stream area of Samchok-gun, eastern costal Kangwon-do, the infected fish were Plecoglossus altivelis and Tribolodon hakonensis. The rats and dogs are infected with the metcercariae obtained from Zacco platypus and Opsariichthys bidens, adult worms collected were Miyata type of Metagonimus with some M. takahashii. When infected with metacercariae from Plecoglossus altivelis, Metagonimus yokogawai was only found. M. yokogawai and Metagonimus Miyata type were found together in Tribolodon hakonensis in Ohsip stream area of Samchok, in the eastern Kangwon-do. The intestinal flukes of genus Metagonimus in western Kangwon-do were Miyata type of Metagonimus and M. takahashii, transmitting mainly by Zacco platypus and Opsariichthys bidens as a source of infection. In the eastern part of Kangwon province (Ohsip stream area of Samchok), M. yokogawai was mainly distributed by P. altivelis and T. hakonesis, but some T. hakonensis harbored the metacercariae of Miyata type of Metagonimus with those of M. yokogawai.

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Digenean parasites of the bivalve mollusc Pisidium amnicum in a small river in eastern Finland.

The host-parasite relationship between digeneans and a semelparous population of the mollusc. Pisidium amnicum Müller in a small river in eastern Finland was studied during 1992/1993. The parasite prevalence of the population was high. The total prevalence was 45.6% in 1992 (n = 790) and 47.5% in 1993 (n = 160). The dominant digenean, Bunodera luciopercae (34.2% in 1992, 35.0% in 1993), had highest prevalences in July/August and in winter. Two other species, Palaeorchis crassus (7.8% in 1992, 7.5% in 1993) and Phyllodistomum elongatum (4.7% and 5.0%), were rare during the winter. The prevalence of B. luciopercae increased as clams aged, while the other species were most common in middle-sized clams. Apparently B. luciopercae rediae dominate over P. elongatum, which has only sporocyst stages, while P. crassus, which has large rediae, is more deleterious to the clam and induces host mortality. Double infections were significantly less common (1.2%) than might be expected by chance. All parasites castrated their hosts; no clam containing both parasites and embryos was found. Semelparity of the population is apparently caused by parasitic castration.

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Fasciolopsiasis: a first case report from Malaysia.

Fasciolopsiasis is a disease caused by the largest intestinal fluke, Fasciolopsis buski. The disease is endemic in the Far East and Southeast Asia. Human acquires the infection after eating raw freshwater plants contaminated with the infective metacercariae. There has been no report of fasciolopsiasis either in man or in animal in Malaysia. We are reporting the first case of fasciolopsiasis in Malaysia in a 39-year-old female farmer, a native of Sabah (East Malaysia). This patient complained of cough and fever for a duration of two weeks, associated with loss of appetite and loss of weight. She had no history of traveling overseas. Physical examination showed pallor, multiple cervical and inguinal lymph nodes and hepatosplenomegaly. Laboratory investigations showed that she had iron deficiency anemia. There was leukocytosis and a raised ESR. Lymph node biopsy revealed a caseating granuloma. Stool examination was positive for the eggs of Fasciolopsis buski. The eggs measure 140 x 72.5 microm and are operculated. In this case, the patient did not present with symptoms suggestive of any intestinal parasitic infections. Detection of Fasciolopsis buski eggs in the stool was an incidental finding. She was diagnosed as a case of disseminated tuberculosis with fasciolopsiasis and was treated with antituberculosis drugs and praziquantel, respectively.

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Fish monogeneans from a shallow, eutrophic Oświn Lake in Poland.

BACKGROUND: A study was carried out in a shallow, eutrophic Oświn Lake, within Seven Islands Reserve (north-eastern Poland), in order to determine the infection of fish with monogeneans as sensitive indicators of water quality. This paper presents comparative analysis of the occurrence of Monogenea in fishes from two, distinctly separated pools of the lake, differing in environmental conditions. The impact of fish size and sampling season on the infection parameters was also analyzed. MATERIAL AND METHODS: In 1998 and 1999, a total of 1091 fish representing 8 dominant species were examined. Samples were collected four times a year (in May, July, August, and October) simultaneously from the eastern and western parts of the lake. RESULTS: 16 species of Monogenea (and some unidentified specimeus) occurred in the fish examined, most of them from the Dactylogyridae family: Dactylogyrus sphyrna, D. auriculatus, D. intermedius, D. anchoratus, D. falcatus, D. tincae, D. wunderi, D. zandti, D. difformis, D. nanus, D. distinguendus, D. crucifer, D. caballeroi. Furthermore, Tetraonchus monenteron (Tetraonchidae), Gyrodactylus elegans (Gyrodactylidae), and Paradiplozoon megan (Diplozoidae) were detected. Monogenea occurred abundantly on the gills of pike, roach, rudd, white bream, carp bream and crucian carp, but rather sporadically in tench and perch. The highest species variety and diversity of monogenean guilds were detected in carp bream and roach. It was proved that D. crucifer and D. difformis were significantly more abundant in the western pool compared to the eastern one. Infection of fish with Dactylogyridae was season-dependent, contrary to the infection of pike with T. monenteron. Intensity of infection of white bream with D. sphyrna and roach with D. crucifer increased with fish body length; such a relationship did not appear for the other monogeneans and their hosts. CONCLUSION: Presence of common Monogenea in the fish examined, lack of rare species, poor diversity of monogenean guilds and relatively low infection rate could be connected with the low stability of the lake environment. Location-dependent occurrence of D. crucifer and D. difformis indicated that the exchange between fish groups from the eastern and western parts of the lake is limited.

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A western blot and ELISA assay for the diagnosis of Taenia solium infection in pigs.

An enzyme-linked immunoelectrotransfer blot (EITB) was developed using soluble Taenia solium metacestode antigen preparation for immunodiagnosis of porcine cysticercosis and compared with an enzyme-linked immunosorbent assay (ELISA). Sera from 20 pigs with parasitologically confirmed cysticercosis were tested by both EITB and ELISA assays. Specificity of the tests was examined by testing 25 serum samples from the UK, where cysticercosis is not endemic and sera from pigs with Echinococcus granulosus (15), Fasciolopsis buski (six), or Trichinella spiralis (five) infections. All but two of the samples from cases of confirmed cysticercosis were positive by EITB and none of the samples from healthy controls or heterologous infections reacted to any of the diagnostic bands. Thus, the test was 90% sensitive and 100% specific. The sensitivity of the ELISA was 70% with 73% specificity, cross-reactions occurring with sera from E. granulosus infected pigs. Four polypeptides (8, 11, 16 and 23 kDa) were identified by SDS-PAGE and EITB that were specifically recognized by pigs with confirmed cysticercosis.

Animals↗

Epidemiological studies of amphistome infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe.

During the period between January 1999 and December 2000, the distribution and seasonal patterns of amphistome infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe were determined through monthly coprological examination. Cattle faecal samples were collected from 12 and nine dipping sites in the highveld and lowveld communal grazing areas, respectively. Patterns of distribution and seasonal fluctuations of intermediate host-snail populations and the climatic factors influencing the distribution were also determined by sampling at monthly intervals for a period of 24 months (November 1998 to October 2000) in six dams and six streams in the highveld and in nine dams in the lowveld communal grazing areas. Each site was sampled for relative snail density and the vegetation cover and type, physical and chemical properties of water, and mean monthly rainfall and temperature were recorded. Aquatic vegetation and grass samples 0-1 m from the edges of the snail habitats were collected monthly to determine the presence or absence of amphistome metacercariae. Snails collected at the same time were individually checked for the emergence of larval stages of amphistomes. A total of 16,264 (calves 5418, weaners 5461 and adults 5385) faecal samples were collected during the entire period of the study and 4790 (29.5%) of the samples were positive for amphistome eggs. For both regions the number of animals positive for amphistome eggs differed significantly between the 2 years, with the second year having a significantly higher prevalence (P < 0.01) than the first year. Significantly higher prevalences were found in the highveld compared to the lowveld (P < 0.001), for adult cattle than calves (P < 0.01), and in the wet over the dry season (P < 0.01). Faecal egg output peaked from October to March in both years of the study. Bulinus tropicus, Bulinus forskalii and Biomphalaria pfeifferi were recorded from the study sites. The main intermediate host for amphistomes was B. tropicus with a prevalence of infection of 8.5%. However, amphistome cercariae were also recorded in Biom. pfeifferi and B. forskalii. Amphistome cercariae were recorded from both the highveld and lowveld areas with peak prevalence during the post-rainy season (March to May). Metacercariae were found on herbage from the fringes of the snail habitats between February and August, with most of the metacercariae concentrated on herbage 0-1 m from the edges of the habitats. Based on the epidemiological findings a control programme was devised. From this study, large burdens of immature flukes could be expected in cattle during the dry months. Since adult cattle would be resistant to the pathogenic effects of the migrating immature amphistomes the target for control would be young animals being exposed to the infection for the first time. Therefore, the first anthelmintic treatment can be administered in calves in mid June when maximum migration of immature amphistomes starting 3-4 weeks after infection in the early dry season would be expected. A second treatment could be given in late July or early August to remove potentially dangerous burdens of immature flukes acquired later in the dry season. Where resources permit, another strategy would be to treat against the mature flukes in March or April in order to reduce the number of eggs deposited on pastures and the opportunity for infection of the intermediate host snails. To reduce cercarial shedding by the intermediate host snails molluscicides can also be applied during the peak transmission periods (April/May and August/September).

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