Main Intermediate Host Research Articles

An optimized DNA extraction and multiplex PCR for the detection of Fasciola sp. in lymnaeid snails

This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex® DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500–600bp) that act as an internal control and a 124bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.

The life cycle of Bucephalus margaritae Ozaki & Ishibashi, 1934 (Digenea, Bucephalidae) from the coast of Santa Catarina State, Brazil

The orange disease is considered the main parasitosis in Perna perna mussel. It is caused by a complex life cycle bucephalid, involving three hosts, among them mollusks and fishes. With the aim of contributing to the knowledge of orange disease in mussel culture, the parasite life cycle was investigated. Experimental studies and monthly samples in the study area allowed the identification and characterization of the Bucephalus margaritae life cycle. Larvae and adults of B. margaritae were fixed in 5% formaldehyde, stained with Gomori’s trichrome, clarified in creosote and mounted in Canada balsam. The cercariae are found in the first intermediate host P. perna inside the sporocysts, which have the form of orange and ramified filaments. The metacercariae encysts in the gills and gill cavity of the second intermediate host, the blenniid Hypleurochilus fissicornis . The definitive host Menticirrhus americanus is infected ingesting blenniids parasitized with metacercariae. The high parasitological indexes of B. margaritae suggests that M. americanus and H. fissicornis act as the main definitive and intermediate hosts, respectively, in the trematode life cycle. The blenniid H. fissicornis is a new intermediate host to the species.

Occurrence of parasitism by Dioctophyma renale in ring-tailed coatis (Nasua nasua) of the Tiete Ecological Park, São Paulo, Brazil

Dioctophymosis is a worldwide renal parasitosis caused by the Dioctophyma renale nematode, which results in progressive destruction of renal tissue. Aquatics annelids are considered the main intermediate hosts and the literature refers as permanent hosts of dogs, wild mammals and even humans. During procedures for population control of coatis (Nasua nasua) in the Ecological Park of Tietê (PET), was noticed the presence of parasitosis by D. renale. From 68 animals, males and females, young and adults, submitted to exploratory laparotomy, 51 were positive for the presence of worms, 9 were found only in the right kidney. In 10 cases, in addition to right kidney parasitism, worms were also observed in the abdominal cavity. In 24 cases D. renale was found only in the abdominal cavity and in 8 animals the right kidney was reduced to a small rigid structure. The study showed that the preferred site for parasitism of the worm, considered erratic, was the abdominal cavity in 66.66% of the cases.

Copepods as the first intermediate hosts of Ligula intestinalis L.: Parasite of bream Abramis brama L. in the Vistula Lagoon of the Baltic Sea

Seven copepod species (potential intermediate hosts of Ligula intestinalis L.) are recorded in the Vistula Lagoon. Megacyclops viridis (Jurine) and Diacyclops bicuspidatus (Claus) are dominant species among the Cyclopoida group and may be the main potential first intermediate host of L. intestinalis. Cytopoidae is unevenly distributed in the lagoon. The parts of the lagoon with high crustacean densities and high levels of bream infestation coincide. The peaks of Cyclopoidae abundance and the highest infestation of the bream by L. intestinalis were recorded in 1998–2000. This study shows that bream infestation in the Vistula Lagoon is directly connected with the presence of freshwater copepods (the first intermediate hosts of L. intestinalis), its abundance, and its spatial distribution over the lagoon area.

Enzootic Sparganosis in Guangdong, People’s Republic of China

To the Editor: Sparganosis is a worldwide parasitic zoonosis caused by infection with spargana, the plerocercoid larvae of various diphyllobothroid tapeworms belonging to the genus Spirometra (1–3). Sparganosis poses a serious threat to human health; the spargana invade mainly the brain, eye, abdominal cavity, spinal cord, and subcutaneous tissues; can damage local tissues; and can cause blindness, paralysis, and even death (4,5). In the People’s Republic of China, sparganosis has emerged as an important foodborne parasitic disease, with ≈1,000 human cases reported in 22 provinces during 1927–2007. Guangdong Province has the most cases (6). Persons in Guangdong Province eat frog meat and place frog poultices made from raw frog meat on open wounds and lesions, which facilitates human infection with spargana. To assess the risk for human infection with sparganosis in this province and to strengthen public food safety awareness, we conducted a comprehensive investigation of spargana infection in frogs, the second intermediate host of Spirometra. By necropsy we examined for spargana 544 frogs (446 Rana nigromaculata and 98 R. tigrina) from Yunfu, Maoming, and Zhanjiang in western Guangdong Province during October 2007–October 2008 (7). Of these 544 frogs, 455 were wild, and 89 were aquacultured. Spargana were found in 27.3% (124/455) of examined wild frogs; of these, 30.0% (107/357) were R. nigromaculata, significantly more (p<0.05) than the 17.3% (17/98) that were R. tigrina. This finding suggests that R. nigromaculata is the main intermediate host of Spirometra in western Guangdong Province. We found 719 spargana in infected wild frogs. The number of worms per frog ranged from 1 to 41, with an average of 5.8 worms per infected frog. No spargana were found in 89 aquacultured R. nigromaculata frogs. The examined wild frogs looked normal and healthy and had no obvious symptoms. During necropsy, we detected local edema, muscle bleeding, and fragile tissues in the tissues invaded by spargana. We also found cysts in some tissues that contained 1 or a few worms. Spargana dissected from host tissue were flat, white worms, which continuously crept in the normal saline. These worms ranged from 2 mm to 115 mm long and from 1 mm to 2 mm wide. Frogs are the second intermediate hosts of Spirometra spp.; pigs, mice, and humans become infected as paratenic hosts by ingesting Spirometra larvae in cyclops or frogs (8,9). Because persons in Guangdong Province enjoy eating frog meat, particularly from wild frogs, many frogs have been sold in the market, including a substantial number of wild frogs. The results of our survey show that infection of wild frogs with spargana reached 27.3% in western Guangdong Province; hence, consumption of wild frogs (and use as poultices) poses a high risk for sparganum infection. Therefore, public health officials, epidemiologists, medical practitioners, parasitologists, veterinarians, and the general public should be aware of such risks and should implement strategies to reduce or eliminate them.

Prevalence of the dog nematode &lt;i&gt;Spirocerca lupi&lt;/i&gt; in populations of its intermediate dung beetle host in the Tshwane (Pretoria) Metropole, South Africa

Spirocerca lupi (Spirurida: Spirocercidae) is a cosmopolitan parasite, principally of domestic dogs and dung beetles are its main intermediate hosts. In South Africa there has recently been growing concern over the upsurge of reported cases of clinical spirocercosis in dogs, while little is known or understood about the dynamics of the host-parasite associations between dung beetles and this nematode. We determined and compared the prevalence of infection in dung beetles between rural, urban and peri-urban areas of Tshwane (Pretoria) Metropole. Dung beetles were sampled during April and October 2006, at various localities in each of these areas. Localities were selected on the basis of being focal areas of high infection with S. lupi in dogs. Pig, dog and cow dung-baited pitfall traps were used for sampling the beetles. Trap contents were collected 48 h after the traps had been set and only dung beetles were collected from the traps. In total, 453 specimens belonging to 18 species were collected from 63 pitfall traps in all three areas. The numbers of species that were collected varied among the three areas. Dung beetles, irrespective of species (18) and numbers (447), predominantly preferred pig dung. The prevalence of dung beetles infected with the larvae of S. lupivaried considerably in the three areas. In the urban area 13.5% of the dung beetles dissected were infected, while the prevalence of S. lupi in dung beetles in the rural area was 2.3%. All the dung beetles that were infected with this nematode showed a preference for omnivore (pig and dog) dung.

Echinococcosis in China, a Review of the Epidemiology of Echinococcus spp.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are highly significant infectious diseases occurring worldwide and caused by metacestodes of tapeworms Echinococcus granulosus and E. multilocularis, respectively. Both human CE and AE have highest prevalence rates in western and northwestern China. Livestock is the main intermediate host of E. granulosus, and wild small mammal are the main intermediate hosts of E. multilocularis. Since they range freely in pastoral areas, prey on wild small mammals and offal of livestock after slaughter, and have close relationships with humans, domestic dogs are the most important definitive host of both Echinococcus spp. with the highest risk of transmitting CE and AE to humans. Pastoralism is the occupation with the highest risk of being infected with the both kinds of echinococcosis due to the proximity of livestock, dogs, and wildlife host species. In this review, we summarize the epidemiology of human echinococcosis, the situation of parasite transmission in animal hosts, and possible transmission patterns in China. In addition, human activities and their potential influence on the transmission of echinococcosis are also discussed.

Infection of foxes byEchinococcocus multilocularisin urban and suburban areas of Nancy, France: influence of feeding habits and environment

This study evaluated the impact of biological and environmental factors on the infection of red foxes (Vulpes vulpes) by Echinococcus multilocularis in an endemic area of north-east France. From January 2004 to April 2006, 127 foxes were examined for E. multilocularis and their stomach contents analysed. The effect of year, season, age, sex and urbanisation level on E. multilocularis presence was estimated using a General Linear Model (GLM) with logit link, (i.e. logistic regression). Urbanisation level was the only influencing factor, with a decreasing gradient from rural [54%, CI 95% (40-68)] to peri-urban [31%, CI 95% (15-52)] and urban area [4%, CI 95% (0.7-15)]. The consumption of Arvicola terrestris and Microtus sp., grassland species, the main presumed intermediate hosts of E. multilocularis, was studied by the same approach. The two species were consumed less in the urban area and more in autumn than in spring. Anthropogenic food consumption was linked to urbanisation and to age. The frequency of anthropogenic food consumption decreased in the rural area. A global model explaining the presence of E. multilocularis and including urbanisation level and diet was then elaborated. Independently of urbanisation, there was a suggestion of less E. multilocularis infection with anthropogenic food consumption. Red foxes consuming Microtus sp. and A. terrestris had higher worm burden than those that did not. The results suggest that the decreasing gradient observed from rural to urban area is linked to behaviour and feeding habits.

The invasive round goby Apollonia melanostoma (Actinopterygii: Gobiidae) - a new intermediate host of the trematode Neochasmus umbellus (Trematoda: Cryptogonimidae) in Lake Erie, Ohio, USA

The invasive round goby Apollonia melanostoma (Actinopterygii: Gobiidae) - a new intermediate host of the trematode Neochasmus umbellus (Trematoda: Cryptogonimidae) in Lake Erie, Ohio, USA

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Bayesian spatial analysis and disease mapping: tools to enhance planning and implementation of a schistosomiasis control programme in Tanzania

To predict the spatial distributions of Schistosoma haematobium and S. mansoni infections to assist planning the implementation of mass distribution of praziquantel as part of an on-going national control programme in Tanzania. Bayesian geostatistical models were developed using parasitological data from 143 schools. In the S. haematobium models, although land surface temperature and rainfall were significant predictors of prevalence, they became non-significant when spatial correlation was taken into account. In the S. mansoni models, distance to water bodies and annual minimum temperature were significant predictors, even when adjusting for spatial correlation. Spatial correlation occurred over greater distances for S. haematobium than for S. mansoni. Uncertainties in predictions were examined to identify areas requiring further data collection before programme implementation. Bayesian geostatistical analysis is a powerful and statistically robust tool for identifying high prevalence areas in a heterogeneous and imperfectly known environment.

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).

Mediterranean Diplodus annularis (Teleostei: Sparidae) and its brain parasite: Unforeseen outcome

Patterns of parasite load and aggregation of the bird trematode Cardiocephaloides longicollis in its main intermediate host in the Mediterranean, the annular sea bream, Diplodus annularis, were studied in a large sample collected off Valencia (Spain) and are discussed within the context of the parasite induced host mortality hypothesis. The metacercariae were located within large composite cysts of host origin in the ventricles of the optic lobes of the cerebrum. A weak immunological response was detected in older fish, which was significantly associated with the total parasite load. Although the mean abundance of C. longicollis showed a tendency to increase with host size, the infection levels were generally homogeneous with a noticeable plateauing in the intermediate size classes. The distribution of the metacercariae was aggregated and agreed with the negative binomial distribution. There was a marked decline in parasite aggregation in the largest size-class, suggesting parasite-induced host mortality in the oldest fish possibly due to predation by large non-host fish predators. On the other hand, levelling off of abundance and decrease in heterogeneity of parasite distribution within the intermediate age cohort could indicate that these sizes are being rapidly and/or constantly removed from the host population due to by-catch fishing. The overall high infection levels and the continuous recruitment across age cohorts provides evidence that an enhanced parasite transmission is taking place in the Gulf of Valencia due to increased spatial overlap of the hosts involved in the life cycle. We suggest a human-induced facilitation of the digenean life cycle due to the fact that gulls in the area feed extensively on discards, thus indicating the possibility of an unforeseen effect of fishing practices in a marine littoral system.

Larval trematode infections in freshwater snails from the highveld and lowveld areas of Zimbabwe.

Between November 1998 and October 2000, freshwater snails were collected monthly from the highveld and lowveld areas of Zimbabwe to determine the occurrence of larval trematodes. A total of 13,789 snails, representing ten species, were collected from 21 sites and 916 (6.6%) harboured patent trematode infections. Eight morphologically distinguishable types of cercariae were identified. Bulinus tropicus had the highest overall prevalence of infection (13.1%). The echinostome was the most common type of cercaria recovered, contributing 38.2% of all infections. Schistosoma cercariae were recovered mainly from the highveld and comprised 8.0% of all infections. Amphistome cercariae contributed 37.6% of all infections and were recorded from both the highveld and lowveld areas with a peak prevalence occurring during the post-rainy period (March-May). The main intermediate host for amphistomes was B. tropicus. Infections in B. globosus, B. forskalii and Biomphalaria pfeifferi with amphistome cercariae are new records for Zimbabwe.

Genetic differentiation, dispersal and mating system in the schistosome-transmitting freshwater snail Biomphalaria glabrata.

Biomphalaria glabrata is the main intermediate host of Schistosoma mansoni in America and one of the most intensely studied species of freshwater snail, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in populations from three regions (Lesser Antilles, Venezuela and southern Brazil). Considerable genetic variation was detected, with an average (s.d.) H(0) = 0.32 (0.24). More diversity per population was found in the Valencia lake basin in Central Venezuela, which suggests an influence of dispersal (via inter-population connectivity) on the restoring of genetic diversity after the demographic bottlenecks recurrently experienced by populations. A marked population structure was detected and there seems to be a relationship between mean differentiation and genetic diversity within regions. There is also a significant isolation-by-distance pattern. The Lesser Antilles populations appear clearly differentiated from the rest, which suggests a single colonisation event followed by local radiation within these islands or multiple colonisation events from the same source area. Our results indicate that B. glabrata essentially cross-fertilises, with little variation in selfing rates among populations. However, significant deficits in heterozygotes and linkage disequilibria were detected in two Venezuelan populations suggesting a mixture of at least two different genetic entities, probably with differences in their respective mating systems.

Fine-scale population structure and dispersal in Biomphalaria glabrata, the intermediate snail host of Schistosoma mansoni, in Venezuela.

Biomphalaria glabrata is the main intermediate host of Schistosoma mansoni in America and one of the most intensely studied species of freshwater snails, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in the Valencia lake basin, which represents the core of the endemic area for schistosomiasis in Venezuela. Populations were sampled at short spatial scale (a few kilometres), both inside the lake and in ponds or rivers near the lake. Our results indicate that B. glabrata essentially cross-fertilizes, with little variation in selfing rates among populations. Our markers detected considerable genetic variation, with an average heterozygosity of 0.60. More diversity per population was found within than outside the lake, suggesting an influence of connectivity among populations on the levels of genetic diversity. A marked population structure was detected and lake populations were less structured than other populations. Most individuals were assigned to their population of origin using an assignment test. No strong demographic signal (e.g. bottleneck) was detected, though lake populations are likely to experience bottlenecks more frequently than the other populations analysed. Differences in gene flow therefore seem to play an important role in population differentiation and in the restoring of genetic diversity in demographically unstable populations.

Genetic variability of the main intermediate host of the Schistosoma mansoni in Brazil, Biomphalaria glabrata (Gastropoda: Planorbidae) assessed by SSR–PCR

The genetic variability of Brazilian Biomphalaria glabrata populations was studied using SSR–PCR. This technique is a variant of the polymerase chain reaction (PCR), which consists of using a single primer directed towards microsatellite regions under high stringency reaction conditions. Twenty snails of each population from eight distant Brazilian localities were analyzed. Morphology and PCR–RFLP were used for previous specific identification of the snails. Bands generated after gel electrophoresis of the SSR–PCR products of each snail were used to study intra- and interpopulation genetic variability. Fifty-five prominent bands were considered in a pairwise band comparison for the determination of genetic variability. Genetic variability was greater between populations than within populations. Snail populations from the field and the laboratory presented almost no genetic differences. No relationship between genetic variability and geographic distance was found. SSR–PCR proved to be a good alternative molecular tool for the population study of B. glabrata.

Report on the occurrence of Angiostrongylus costaricensis in southern Brazil, in a new intermediate host from the genus Sarasinula (Veronicellidae, Gastropoda).

Veronicellid slugs are the main intermediate hosts for Angiostrongylus costaricencis. In a rural locality in Nova Itaberaba (SC, southern Brazil) Sarasinula linguaeformis was identified as a crop pest. The parasitological examination revealed A. costaricencis infection in 43 out ot 50 slugs. The prevalence of 86% and the individual parasitic burdens are the highest sofar reported in Brazil and S. linguaeformis is the first species from the genus Sarasinula to be identified as intermediate host for A. costaricencis in southern Brazil.

Angiostrongylus costaricensis and the intermediate hosts: observations on elimination of L3 in the mucus and inoculation of L1 through the tegument of mollucs.

Human accidental infection with Angiostrongylus costaricensis may result in abdominal disease of varied severity. Slugs from the Veronicellidae family are the main intermediate hosts for this parasitic nematode of rodents. Phyllocaulis variegatus, Phyllocaulis soleiformis and Phyllocaulis boraceiensis were experimentally infected to describe the kinetics of L3 elimination in the mucus secretions of those veronicelid species. A maximum of 2 L3/g/day was found in the mucus, while the number of L3 isolated from the fibromuscular tissues varied from 14 to 448. Productive infection was established by inoculations in the hyponotum or in the body cavity, through the tegument. Intra-cavity injection is a less complex procedure and permits a better control of inocula. A preliminary trial to titrate the infective dosis for P. variegatus indicated that inocula should range between 1000 and 5000 L1. The data also confirmed the importance of P. variegatus as an intermediate host of A. costaricensis.

Efficacy of Selected Compounds on Thermocyclops oblongatus, one of the Main Intermediate Hosts of Guinea Worm in Africa

AbstractTen selected compounds were tested on Thermocyclops oblongatus to determine their ability to be used for treatment of drinking water sources. None were shown to be interesting for individual application in household water tanks. Tested compounds, such as potassium permanganate or chlorine‐releasing products, were ineffective or affected dramatically the taste or colour of the water. For collective application in water sources, temephos appeared to be the most valuable pesticide because it was effective against cyclops and harmless for environment. Other pesticides should be proposed as substitutes in case of cyclops resistance to temephos.