Comparative Phylogenetic Studies on Schistosoma japonicum and Its Snail Intermediate Host Oncomelania hupensis: Origins, Dispersal and Coevolution.

BACKGROUND: Schistosoma japonicum causes major public health problems in China and the Philippines; this parasite, which is transmitted by freshwater snails of the species Oncomelania hupensis, causes the disease intestinal schistosomiasis in humans and cattle. Researchers working on Schistosoma in Africa have described the relationship between the parasites and their snail intermediate hosts as coevolved or even as an evolutionary arms race. In the present study this hypothesis of coevolution is evaluated for S. japonicum and O. hupensis. The origins and radiation of the snails and the parasite across China, and the taxonomic validity of the sub-species of O. hupensis, are also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The findings provide no evidence for coevolution between S. japonicum and O. hupensis, and the phylogeographical analysis suggests a heterochronous radiation of the parasites and snails in response to different palaeogeographical and climatic triggers. The results are consistent with a hypothesis of East to West colonisation of China by Oncomelania with a re-invasion of Japan by O. hupensis from China. The Taiwan population of S. japonicum appears to be recently established in comparison with mainland Chinese populations. CONCLUSIONS/SIGNIFICANCE: The snail and parasite populations of the western mountain region of China (Yunnan and Sichuan) appear to have been isolated from Southeast Asian populations since the Pleistocene; this has implications for road and rail links being constructed in the region, which will breach biogeographical barriers between China and Southeast Asia. The results also have implications for the spread of S. japonicum. In the absence of coevolution, the parasite may more readily colonise new snail populations to which it is not locally adapted, or even new intermediate host species; this can facilitate its dispersal into new areas. Additional work is required to assess further the risk of spread of S. japonicum.

High infectivity of Toxocara cati larvae from muscles of experimentally infected rats.

The organ distribution of Toxocara cati larvae in albino rats Rattus norvegicus (n=6/group) experimentally inoculated with 1000 embryonated eggs was examined 1, 2, 3, 7, 30, 90, and 180 days post inoculation (dpi), and the infectivity of recovered larvae was evaluated by bioassay in mice. The intestines, liver, lungs, muscles (carcass) and other organs (heart, brain, spleen, kidneys and genital organs) were digested for larval recovery. Larvae were recovered from all rats, with the mean number of recovered larvae ranging from 13.3 at 1 dpi to 135.6 at 90 dpi. Most of the larvae recovered were detected in the intestines (56.3%) and liver (43.8%) at 1 dpi; liver (21.6%) and lungs (69.6%) at 2 dpi; muscles (45.9%) and lungs (36.9%) at 3 dpi. Subsequently, most of larvae were recovered from muscles at 7 dpi (92.5%), 30 dpi (97.8%), 90 dpi (99.4%) and 180 dpi (99.1%). In the mouse bioassay, 43.8% of 90-day-old larvae and 43.0% of 180-day-old larvae recovered from rats established in mice. The present study demonstrated that T. cati larvae persist predominantly in rat muscles and nearly half of them retain infective for at least half a year. The results indicate that R. norvegicus may be a suitable paratenic host of T. cati under natural conditions.

Tolerance to low temperatures of Toxocara cati larvae in chicken muscle tissue.

Infectivity of Toxocara cati larvae in muscle tissue of chickens after storage at 4°C and -25°C was assessed in a mouse bioassay to provide information on the risk of meat-borne toxocarosis. Muscle tissue samples of 30-day old T. cati infections were stored at 4°C for 14 and 28 days and at -25°C for 12, 24 and 48 h, whereafter, larvae were released by digestion. For each experimental group, the released larvae were inoculated in six mice. After 15 days, mice were euthanized and larval burden was assessed by digestion. In the control group (no storage of the infected chicken meat), 47.9% of the inoculated larvae established in mice, whereas storage of meat at 4°C for 14 days or 28 days reduced the recovery to 24.1% or 3.3%, respectively. Muscle larvae exposed to -25°C for 12, 24 or 48h did not establish in the mice. The observation that larvae retain infective after refrigeration at exposure in 4°C for 28 days, emphasize the zoonotic potential of poultry meat as a causative agent of human toxocarosis.

Genetic structure of Ascaris roundworm in Japan and patterns of its geographical variation.

Ascaris roundworm isolates from Japan and central Europe were examined by sequencing analyses to better understand geographically induced nucleotide variation and genotype distribution according to host. Three well-supported clusters (denoted as A, B, C) were identified by generating cox1 sequences of mtDNA from these regions. Among 5 pig isolates collected in eastern Honshu, Japan, in 2010, 3 carried DNA characteristics for cluster A and 2 corresponded with the characteristics of cluster B. The sequence of the human isolate JH1 from north-central Honshu, fixed in formalin since 1972, conformed to the characteristics of cluster A. Differential analysis of ribosomal ITS1 region revealed the JH1 isolate sequence profile of Ascaris lumbricoides. Cluster C, which was the most distinguish cluster, was formed by reference Slovak isolates and has been so far found almost exclusively in European pigs. A fluctuating prevailing distribution of A and B lineages in human and pig hosts in different territories of the world and the global distribution of several haplotypes indicate their establishment before secondary differentiation in a given region due to host affiliation. The protocol established for DNA isolation from formalin-fixed specimens using the modified procedure with the Qiagen extraction set can be used as a tool for retrospective studies in ascarid helminths when only archival specimens are available.

Toxocara cati larvae persist and retain high infectivity in muscles of experimentally infected chickens.

The distribution of Toxocara cati larvae in the organs of chickens (n=31), experimentally inoculated with 3000 embryonated eggs, was examined 1, 2, 3, 7, 29, 86 and 175-176 days post-infection (dpi), and the infectivity of recovered larvae was evaluated by bioassay in mice. The duodenum, liver, lungs, heart, brain, pectoral muscles (white meat), and hindlimb muscles (red meat) of the chickens were HCl-pepsin digested for larval recovery. Larvae were recovered from all chickens [mean=220.4 ± 114.9 (SD)], and although no decrease of total larval recovery was observed over time, predilection sites changed: liver (92.6% of larval recovery) at 1 dpi; lungs (77.3%) and liver (20.9%) at 2 dpi; lungs (80.8%), muscles (9.4%), and liver (8.5%) at 3 dpi; muscles (52.0%) and lungs (45.6%) at 7 dpi, whereafter most larvae were recovered from muscles: 29 dpi (99.5%), 86 dpi (99.3%) and 175-176 dpi (99.6%). In the bioassay, 52.9% of larvae from 175 to 176 days old infections in chickens established in mice, which demonstrates that T. cati larvae retain infective in the muscles of chickens for half a year. These results highlight the zoonotic potential of poultry meat as a causative agent of human toxocarosis.

Spatial analysis and remote sensing for monitoring systems of Oncomelania nosophora following the eradication of schistosomiasis japonica in Yamanashi Prefecture, Japan.

In order to develop an inexpensive, simple, and accurate method of monitoring for the reemergence of schistosomiasis japonica in Yamanashi Prefecture, Japan, the distribution and habitation density of the intermediate host, Oncomelania nosophora, were spatially analyzed using geographic information systems. The 1967-1968 density distribution maps prepared by Yamanashi Prefecture and Nihei were digitized and geocoded. The habitats and population density of O. nosophora were estimated by referring to the data compiled by the Yamanashi Association for Schistosomiasis Control (1977). These earlier findings were compared with average population densities between 1996 and 2000 previously recorded (Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 52, 395-401, 2003 and Nihei, N., Kajihara, N., Kirinoki, M., et al., Parasitol. Int., 53, 199-205, 2004). A variance map was created to compare the spatial distribution maps of population density from each of the two periods of interest. The changes in distribution were remarkable and the map was found to be effective for future control. The most appropriate monitoring sites were chosen on the basis of the spatial population density maps and the variance map. Moreover, the paddy fields at risk were extracted using the normalized difference vegetation index value based on Advanced Land Observation Satellite images. The combination of this method with the global positioning system provides an inexpensive means of monitoring modern schistosomiasis endemic areas in Japan and also in China, the Philippines, and other countries as well, where the intermediate snail grows in paddy fields and marshlands under consistently wet conditions.

Yeast-generated CO2 as a convenient source of carbon dioxide for adult mosquito sampling.

A new, convenient method was developed to supply CO2 for mosquito sampling by using yeast, which converts sugar into CO2 and ethyl alcohol. The system could, at average, generate 32.4 ml/min of CO2 for at least 27 h. The total weight of the CO2 generated was estimated to be 94 g. The efficacy of yeast-generated CO2 as attractant for mosquitoes was significant, and the following 6 mosquito species were collected using yeast-generated CO2 traps from July to September 2003 in a residential area of southern and northern Yokohama City, Japan: Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), Culex halifaxii Theobald, Cx. pipiens pallens Coquillett, Ochlerotatus japonicus (Theobald), and Tripteroides bambusa (Yamada). Besides mosquitoes, various other insects were collected in the trap. Species compositions of insects collected in yeast-generated CO2 traps and dry-ice-baited traps were compared.

Fixed-point observation of Oncomelania nosophora in Kofu Basin -- establishment of monitoring system of schistosomiasis japonica in Japan.

There are still many Oncomelania snails that inhabit the Kofu Basin, Yamanashi Prefecture, Japan, which had been declared free of schistosomiasis japonica. Due to the need to monitor the situation, a fixed-point observation system using GIS from GPS is being examined. In addition, in broad present or former endemic areas, survey areas are being managed by remote sensing with satellite images or aerial photographs. A simple and effective monitoring method by mobile GIS using PDAs was developed, risk or hazard maps were prepared and a system that would enable a response in the event of reemergence is being examined.

Fixed-point observation of Oncomelania nosophora in Kofu Basin--establishment of monitoring system of schistosomiasis japonica in Japan.

There are still many Oncomelania snails that inhabit the Kofu Basin, Yamanashi Prefecture, Japan, which had been declared free of schistosomiasis japonica. Due to the need to monitor the situation, a fixed-point observation system using GIS from GPS is being examined. In addition, in broad present or former endemic areas, survey areas are being managed by remote sensing with satellite images or aerial photographs. A simple and effective monitoring method by mobile GIS using PDAs was developed, risk or hazard maps were prepared and a system that would enable a response in the event of reemergence is being examined.