Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.     

Avian Influenza Infection Alters Fecal Odor in Mallards

Changes in body odor are known to be a consequence of many diseases. Much of the published work on disease-related and body odor changes has involved parasites and certain cancers. Much less studied have been viral diseases, possibly due to an absence of good animal model systems. Here we studied possible alteration of fecal odors in animals infected with avian influenza viruses (AIV). In a behavioral study, inbred C57BL/6 mice were trained in a standard Y-maze to discriminate odors emanating from feces collected from mallard ducks (Anas platyrhynchos) infected with low-pathogenic avian influenza virus compared to fecal odors from non-infected controls. Mice could discriminate odors from non-infected compared to infected individual ducks on the basis of fecal odors when feces from post-infection periods were paired with feces from pre-infection periods. Prompted by this indication of odor change, fecal samples were subjected to dynamic headspace and solvent extraction analyses employing gas chromatography/mass spectrometry to identify chemical markers indicative of AIV infection. Chemical analyses indicated that AIV infection was associated with a marked increase of acetoin (3-hydroxy-2-butanone) in feces. These experiments demonstrate that information regarding viral infection exists via volatile metabolites present in feces. Further, they suggest that odor changes following virus infection could play a role in regulating behavior of conspecifics exposed to infected individuals.     

Schistosoma mekongi: the in vitro effect of praziquantel and artesunate on the adult fluke

The efficacy and tolerance of 80 microg/ml praziquantel (PZQ) and 40 microg/ml artesunate (ATS) against adult stage Schistosoma mekongi in vitro were investigated after 3, 6, 12, and 24h incubation by monitoring worm motility and compared tegumental changes using scanning electron microscopy (SEM). Thirty mice were infected with S. mekongi cercaria for 49 days. Adult worms were collected by perfusion method and prepared for in vitro study. Contraction and decreased motor activity were observed after as little as 3h incubation with PZQ and ATS. Some of the worms were immobile 12h after exposure, and died within 24h. The tegument of S. mekongi showed severe swelling, vacuolization and disruption, fusion of the tegumental ridges, collapse and peeling. After 12-24h incubation, PZQ induced similar but they less severe, tegumental changes to those observed after exposure to ATS. The direct observation of the fluke motility and SEM study suggest that ATS is more effective than PZQ in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.     

Detection by PCR of the Tyzzer's disease organism (Clostridium piliforme) in feces.

We examined whether the Tyzzer's disease organism, Clostridium piliforme, could be detected in feces by PCR. If the organism could be detected in feces, a diagnosis could be made without sacrifice of the animal. Using the RT strain of C. piliforme, we found that a C. piliforme band could be detected when there were > or = 1 x 10(0) bacteria present in the PCR solution, but the presence of fecal extract in the solution depressed the sensitivity 10 fold. Nevertheless, we could detect the C. piliforme-specific band in fecal extracts from rats in a naturally infected colony, and concluded that the use of PCR to detect C. piliforme DNA in fecal extracts would be a useful diagnostic technique.     

The respiratory properties of Biomphalaria glabrata exposed to Schistosoma mansoni infection,starvation, CO,and choices of different oxygen concentrations

The oxygen consumption rate (VO(2)) of Biomphalaria glabrata populations, using polarometric and manometric methods, when plotted against dried body mass as logarithmic co-ordinates, respectively, fell on a regression line with a slope between 0.933 and 1.02. The slope of the regression line for non-infected Schistosoma mansoni populations was found to be 1.04 with no differences in the VO(2) between infected and non-infected snails. The VO(2) of CO-treated snails was the same as for the control snails. The VO(2) of starved snails declined after 3 days and was half the original value after 10 days starvation at 27 degrees C. The P(50) value for snail haemolymph containing haemoglobin suspended in a Tris-HCl buffer was 5.57(+/-0.73)mmHg at a pH of 7.51 and 25 degrees C. For Sephadex-75 cleaned haemolymph the P(50) value was 1.72(+/-0.07)mmHg at 25 degrees C and pH 7.51. Snails exposed to oxygen fs and to choices of different oxygen concentrations in water did not exclusively prefer high (130mmHg), low (15mmHg), or normal (80mmHg) oxygen tensions. The oxygen consumption rate of 782 cercariae at 27 degrees C was measured as 0.0092 microl O(2)/h per single cercaria. The results, when compared with the data in the literature [Z. Vergl. Physiol. 46 (1963) 467;; S. A. J. Zool. 14 (1979) 202], indicate that the mantle cavity gas bubble plays an insignificant or no role at all when pulmonate snails are kept in water with high partial pressures of oxygen and at low temperatures.     

The pig as a host for Schistosoma mekongi in Laos.

A survey of helminths in domestic pigs was conducted in Khong District, Laos, to elucidate if these domestic animals could act as definitive hosts for Schistosoma mekongi and to obtain a general overview of their helminthological infection status. Fecal samples were collected from 98 pigs. Twelve pigs (12.2%) were found to excrete S. mekongi eggs. Infection was confirmed by detection of S. mekongi eggs in tissues of liver, rectum, and cecum of 2 pigs. A total of 75.8% of the pigs was infected with 1 or more helminth species. This study showed that pigs may act as a definitive host for S. mekongi.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

The potential for false-positive diagnosis of protostrongyliasis by extraction of larvae from feces

The potential of protostrongylid first-stage larvae (L1) to survive passage through the alimentary canal of non-infected mammals was investigated. Parelaphostrongylus tenuis L1 were collected from feces of an experimentally infected white-tailed deer (Odocoileus virginianus). We utilized two red deer (Cervus elaphus) and four laboratory rats (Rattus norvegicus) which were each fed the L1 of P. tenuis. Larvae were recovered, intact and alive, from the fecal samples of all six animals. Larvae of P. tenuis, and probably of other related species, can survive passage through the alimentary canal of uninfected mammals and they can be collected from feces using the Baermann technique and other related larval extraction methods. Rain water was found to be successful in the dispersal of P. tenuis L1 from the feces of infected animals. These findings raise the possibility of ingestion of L1 and their subsequent passage, by uninfected animals. This potential for false-positive diagnosis of infection in live animals necessitates accurate interpretation of a host's infection-status. Such findings reinforce the need for a reliable method of diagnosing infections in live animals.     

Segmentina hemisphaerula: a new molluscan intermediate host for Echinostoma cinetorchis in Korea.

Three species of Planorbidae have been reported from Korea, e.g., Gyraulus convexiusculus, Hippeutis (Helicorbis) cantori, and Segmentina (Polypylis) hemisphaerula. Of these, only H. cantori was reported as the first and second intermediate host for Echinostoma cinetorchis, an important human intestinal parasite in Korea. Segmentina hemisphaerula has also been found to be an intermediate host. In field-collected planorbids, only S. hemisphaerula was found shedding echinostome cercariae and infected with metacercariae of E. cinetorchis, whereas no G. convexiusculus and H. cantori were found to be infected. In experiments with laboratory-bred snails, G. convexiusculus and S. hemisphaerula were susceptible to infection by miracidia of E. cinetorchis, but H. cantori could not be infected. Tadpoles of Rana nigromaculata and laboratory-bred snails of the 3 planorbid species were exposed to E. cinetorchis cercariae shed from field-collected S. hemisphaerula. All tadpoles, S. hemisphaerula, and G. convexiusculus became infected, but no H. cantori were infected. Metacercariae from tadpoles, S. hemisphaerula, and G. convexiusculus were fed to rats per os, and eggs of E. cinetorchis were detected in the rat feces 1 wk later. The rats were killed, and adult E. cinetorchis were recovered from the small intestines. This is the first report of G. convexiusculus as a potential first and second intermediate host and of S. hemisphaerula as a new first and second intermediate host for E. cinetorchis in Korea.     

Comparison of Arcobacter isolation methods, and diversity of Arcobacter spp. in Cheshire, United Kingdom

The aims of this study were, firstly, to compare five published methods for the isolation of Arcobacter spp. from animal feces in order to determine the most sensitive and specific method. Second, we analyzed the resulting isolates by multilocus sequence typing (MLST) in order to investigate the diversity of the isolates recovered. Third, we investigated the ability to recover Arcobacter spp. from frozen fecal samples. Seventy-seven fecal samples from cattle, sheep, and badgers were subjected to five isolation methods, based on published methods for the isolation of Arcobacter and Campylobacter spp. Thirty-nine Arcobacter butzleri isolates were analyzed using a multilocus sequence typing scheme. The survival of Arcobacter spp. in frozen samples was investigated by freezing the fecal samples at -80°C for 7 days and then applying the same five isolation methods. The most sensitive and specific method used an Arcobacter-specific broth in conjunction with modified charcoal cefoperazone deoxycholate agar (mCCDA) with added antibiotics. Freezing of fecal samples led to a reduction in the recovery of Arcobacter spp. by approximately 50%. The 39 allelic profiles obtained by MLST could be divided into 11 sequence types (STs). We have identified the most sensitive and specific method for the isolation of Arcobacter spp. from animal feces and demonstrated that the freezing of fecal samples prior to isolation reduces arcobacter recovery. MLST analysis of the isolates revealed a high level of diversity.     

High susceptibility of Neotricula aperta gamma-strain from Krakor and Sdau in Cambodia to Schistosoma mekongi from Khong Island in Laos

Neotricula aperta gamma-strain snails collected from Krakor and Sdau in Cambodia were found to have the same or higher susceptibility to Schistosoma mekongi as N. aperta originally isolated from Khong in Laos. Infection rates of N. aperta gamma-strain snails exposed to 3 miracidia at week 8 were: Khong gamma-strain, 22.6%; Krakor gamma-strain, 33.3%; and Sdau gamma-strain, 67.4%. At week 10, the Sdau gamma-strain showed the highest infection rate of 83.3%. We thus found significantly high susceptibility of the Sdau gamma-strain to S. mekongi originally isolated from Khong. However, in another experiment, susceptibility of the Sdau gamma-strain was rather comparable to that of Khong and Krakor gamma-strain. We also found no significant differences in infection and survival rates between the Khong and Krakor gamma-strain when the snails were exposed to 3 or 6 miracidia. This is the first report to confirm the high susceptibility in the laboratory of N. aperta gamma-strain snails from endemic areas in Cambodia to S. mekongi originally isolated from Laos. The high susceptibility of N. aperta gamma-strain snails to S. mekongi in distant areas may be an important factor in the endemic transmission of human schistosomiasis.     

Coproantigen capture ELISA for detection of Clonorchis sinensis infection in experimentally infected rats

The present study investigated the diagnostic value of an ELISA for the detection of Clonorchis sinensis antigen in the feces of experimentally infected rats. A mouse polyclonal IgG antibody against adult C. sinensis crude antigen (CsAg) was used to capture the C. sinensis coproantigen. The detection limit for pure CsAg was 20 ng/ml in sample buffer and 40 ng/ml in uninfected fecal extract. The test was evaluated using a follow-up of five groups of rats experimentally infected with 100, 50, 10, 5 and 1 metacercariae of C. sinensis and an uninfected control group. Coproantigen was detected in all infected groups of rats from 2 weeks of infection, whereas fecal eggs were not observed until 3 weeks of infection. As the infection period progressed, the fecal CsAg concentration increased in all groups of infected rats, even those infected with a single metacercaria. The fecal CsAg concentration was correlated positively with fecal egg counts and worm burden. This coproantigen capture ELISA is highly sensitive for the detection of CsAg in rat feces, and with further development, should be useful for mass screening of human subjects in clonorchiasis-endemic areas.     

洞庭湖外睾吸虫新种及其生活史

本文报告洞庭湖区鲶鱼肠道寄生的洞庭湖外睾吸虫Exorchis dongtinghuensis sp.nov(新种)及其全程生活史,其第一中间宿主为湖北钉螺Oncomelania hupensis;第二中间宿主为鲤鱼、鲫鱼和金鱼;终宿主为鲶鱼Parasilurus asotus。作者对各期宿主作了人工感染试验和现场自然感染调查。对其发育过程作了观察比较。     

Fecal bacterial contamination in natural water reservoirs as an indicator of seasonal infection by Opisthorchis viverrini in snail intermediate hosts

Opisthorchis viverrini, a carcinogenic liver fluke, requires Bithynia snails as the first intermediate host, which release cercariae after ingesting fluke eggs from contaminated water. Fecal bacterial contamination and O. viverrini-infected Bithynia snails were investigated in samples collected from natural water reservoirs in Ban Phai, Chonnabot and Muang Districts (Ban Lerngpeuy) in Khon Kaen Province, northeast Thailand, where there is a high incidence of cholangiocarcinoma. Water was sampled and examined six times (February, April, June, August, October and December 2006). The most probable number (MPN) index and coliform counts were utilized to evaluate fecal contamination; the cercarial shedding method was conducted for detecting infected snails. The data revealed that all water samples had a high MPN index number, and fecal coliform levels above the WHO standard. This indicated that water in these reservoirs was contaminated with feces or manure constituents. Water sampling from Ban Lerngpeuy showed full-scale bacterial contamination (> 1609 MPN index) throughout the year. This finding was correlated with the highest prevalence of O. viverrini-infected snails, which were found nearly all year round in this area. Slightly lower fecal contamination levels were detected in water samples from Chonnabot and Ban Phai, with high MPN index numbers and coliform counts from April to October. This corresponded with the higher recovery of infected snails in June and August, but with relatively lower prevalence than those found in Ban Lerngpeuy. Among the sampling sites, the people in Ban Lerngpeuy live nearer to the reservoir than do those in Ban Phai and Chonnabot. These results indicate that fecal bacterial contamination in natural water reservoirs is an important indicator of seasonal transmission of O. viverrini eggs to snail intermediate hosts. Sanitation improvement is essential and future investigations on the sources of contamination are needed.     

Application of nested polymerase chain reaction to detection of mouse hepatitis virus in fecal specimens during a natural outbreak in an immunodeficient mouse colony.

The usefulness of RT-PCR for the detection of MHV in tissues and feces of experimentally infected animals has been reported, but it was unclear whether the method was also applicable for the detection of MHV during a natural outbreak. Enterotropic infection is considered to be the most common form of natural infection among various forms of MHV infection. In this paper, RT-nested PCR was performed to detect MHV excreted in the feces during an outbreak in an immunocompromised A/WySnJ mouse colony. The expected bands were amplified after nested PCR from 20 fecal samples out of 37. These results showed that RT-nested PCR could be applicable for the diagnosis for MHV natural infection.     

Ribeiroia marini: pathogenesis and larval trematode antagonism in the snail, Biomphalaria glabrata

Larval trematode antagonism between Ribeiroia marini and Schistosoma mansoni was studied in the snail Biomphalaria glabrata. A laboratory-raised Puerto Rican strain of B. glabrata was exposed to single and double infections with given numbers of: (1) embryonated eggs of R. marini from laboratory rats, and (2) miracidia of S. mansoni from mice. Snails were maintained in outside environmental tanks in San Juan, Puerto Rico and larval trematode interactions were examined in a series of five experiments. Snails of all sizes were highly susceptible to single infections with R. marini. Rediae and cercariae caused extensive damage to the digestive gland and ovotestis resulting in premature death of snails. Heavily infected snails were castrated and stopped laying eggs. Snails infected first with S. mansoni were only partly susceptible to superinfection with R. marini given on Day 23. In a reverse experiment, snails infected first with R. marini were only partly susceptible to a second infection with S. mansoni given on Day 23. In simultaneous exposures, snails developed double infections (22%) with R. marini dominant and S. mansoni sporocyst and cercaria production reduced. While R. marini is not a strong direct antagonist against established S. mansoni infections, it has several attributes as a possible biological control agent: hardy eggs easily produced in rats; high infectivity to snails of all ages; and ability to castrate and prematurely kill B. glabrata. The R. marini-rat system described here provides a convenient laboratory and field model for the study of intrasnail trematode antagonism and biological control.     

Determination of diaminopimelic acid in rat feces by high-performance liquid chromatography using the Pico Tag method

The purpose of the study was to develop a method for the determination of diaminopimelic acid (DAPA) concentrations in rat feces by reversed-phase high-performance liquid chromatography (HPLC) using the Pico Tag method. Precolumn derivatization with phenylisothyocyanate (PITC) and UV (254 nm filter) detection were used. Samples were hydrolysed in 6 M HCl at 110 degrees C for 24 h. Hydrolysates were then diluted, dried and derivatized, and samples (10 microliter injected onto a 300x3.9 mm NovaPak C(18) (Waters) HPLC column. Under the conditions used, DAPA eluted as one single peak between those of tyrosine and valine. On-column DAPA concentrations in standards were 41.5-83 pmol, which were in the range of the amounts present in fecal samples of rats fed semisynthetic diets. Amounts of DAPA determined in fecal samples of rats fed broad bean- or chickpea-based diets were, respectively, 2.56 and 2.98 mg g(-1). The advantages of the method and the relevance of the results for nutritional studies in monogastric animals are discussed.     

Microbial community analysis and identification of alternative host-specific fecal indicators in fecal and river water samples using pyrosequencing

It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%-52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.