Genetic analyses of Chinese isolates of Toxoplasma gondii reveal a new genotype with high virulence to murine hosts.

A great deal of evidence demonstrates that a strongly clonal population structure of Toxoplasma gondii strains exists in humans and animals in North America and Europe, while the strains from South America are genetically separate and more diverse. Potential differences in virulence between different strains mean that an understanding of strain diversity is important to human and animal health. However, to date, only one predominant genotype, ToxoDB#9 (Chinese I), and a few other genotypes, including ToxoDB#205, have been identified in China. By using DNA sequence-based phylogenetic analyses, we have re-evaluated the population structure of T. gondii strains collected from China and compared them with other global strains. Based on phylogenetic analysis of restriction fragment length polymorphisms, multilocus sequence typing and intron sequences from T. gondii, we propose that the Chinese isolates described as Chinese I are divided into two groups called Chinese I and Chinese III. Our results demonstrate that significant differences were found in mouse mortality caused by some Chinese strains, and also the archetypal I, II, III strains in mice. Furthermore, a comparison of cyst loading in the brains of infected rats showed some Chinese strains to be capable of a high degree of cyst formation. Furthermore we show that genotyping using neutral genetic markers may not be a useful predictor of pathogenic phenotypes.

Prevalence of Toxoplasma gondii in localized populations of Apodemus sylvaticus is linked to population genotype not to population location.

Toxoplasma gondii is a globally distributed parasite infecting humans and warm-blooded animals. Although many surveys have been conducted for T. gondii infection in mammals, little is known about the detailed distribution in localized natural populations. In this study, host genotype and spatial location were investigated in relation to T. gondii infection. Wood mice (Apodemus sylvaticus) were collected from 4 sampling sites within a localized peri-aquatic woodland ecosystem. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii was detected using 4 specific PCR-based markers: SAG1, SAG2, SAG3 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples were positive giving an infection rate of 34.92% (95% CI: 27.14-43.59%). Juvenile, young adults and adults were infected at a similar prevalence, respectively, 7/17 (41.18%), 27/65 (41.54%) and 10/44 (22.72%) with no significant age-prevalence effect (P = 0.23). Results of genetic analysis of the mice showed that the collection consists of 4 genetically distinct populations. There was a significant difference in T. gondii prevalence in the different genotypically derived mouse populations (P = 0.035) but not between geographically defined populations (P = 0.29). These data point to either a host genetic/family influence on parasite infection or to parasite vertical transmission.

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails.

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.

Toxoplasma gondii infection in pregnant women in China.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is one of the most common parasitic infections in humans. Primary infection in pregnant women can be transmitted to the fetus leading to miscarriage or congenital toxoplasmosis. Carefully designed nationwide seroprevalence surveys and case-control studies of risk factors conducted primarily in Europe and America, have shaped our view of the global status of maternal and congenital infection, directing approaches to disease prevention. However, despite encompassing 1 in 5 of the world's population, information is limited on the status of toxoplasmosis in China, partly due to the linguistic inaccessibility of the Chinese literature to the global scientific community. By selection and analysis of studies and data, reported within the last 2 decades in China, this review summarizes and renders accessible a large body of Chinese and other literature and aims to estimate the seroprevalence in Chinese pregnant women. It also reviews the prevalence trends, risk factors, and clinical manifestations. The key findings are (1) the majority of studies show that the overall seroprevalence in Chinese pregnant women is less than 10%, considerably lower than a recently published global analysis; and (2) the few available appropriate studies on maternal acute infection suggested an incidence of 0·3% which is broadly comparable to studies from other countries.

Human Angiostrongylus cantonensis: an update.

Angiostrongylus cantonensis was first discovered in 1935 and has become an important emerging pathogen causing human angiostrongyliasis. Major outbreaks of human angiostrongyliasis have been reported in endemic regions. Thousands of cases of human angiostrongyliasis have been documented worldwide. A. cantonensis has spread from its traditional endemic regions of the Pacific islands and Southeast Asia to the American continent including the USA, Caribbean islands and Brazil. Humans acquire A. cantonensis by consumption of raw or undercooked intermediate snail hosts or paratenic hosts. The main clinical manifestations of human angiostrongyliasis are eosinophilic meningitis and ocular angiostrongyliasis. The treatment of this disease includes supportive treatment, corticosteroid therapy, and combined therapy with corticosteroids and anthelminthics. The most effective method for prevention is to persuade people not to eat raw or undercooked intermediate and paratenic hosts.

Prevalence and co-infection of Toxoplasma gondii and Neospora caninum in Apodemus sylvaticus in an area relatively free of cats.

The protozoan parasite Toxoplasma gondii is prevalent worldwide and can infect a remarkably wide range of hosts despite felids being the only definitive host. As cats play a major role in transmission to secondary mammalian hosts, the interaction between cats and these hosts should be a major factor determining final prevalence in the secondary host. This study investigates the prevalence of T. gondii in a natural population of Apodemus sylvaticus collected from an area with low cat density (<2·5 cats/km2). A surprisingly high prevalence of 40·78% (95% CI: 34·07%-47·79%) was observed despite this. A comparable level of prevalence was observed in a previously published study using the same approaches where a prevalence of 59% (95% CI: 50·13%-67·87%) was observed in a natural population of Mus domesticus from an area with high cat density (>500 cats/km2). Detection of infected foetuses from pregnant dams in both populations suggests that congenital transmission may enable persistence of infection in the absence of cats. The prevalences of the related parasite, Neospora caninum were found to be low in both populations (A. sylvaticus: 3·39% (95% CI: 0·12%-6·66%); M. domesticus: 3·08% (95% CI: 0·11%-6·05%)). These results suggest that cat density may have a lower than expected effect on final prevalence in these ecosystems.

Genotyping of Toxoplasma gondii isolates from cats in different geographic regions of China.

Fourteen isolates of Toxoplasma gondii were isolated from cats from 4 different geographic provinces (Anhui, Hubei, Shanxi and Guangdong) in China and their genetic diversity with 8 nuclear loci SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico, was analysed by restriction fragment length polymorphisms (RFLPs). Two genotypes from these 14 isolates were identified but none of them belongs to the typical genetic types (types I, II and III). It is unexpected that such high similarity was observed in these 14 isolates although their original regions are significantly distant. Our results strongly indicate that the three traditional clonal lineages of types I, II and III of this parasite may not be preponderant in China. In addition, our results show that the genotypes of T. gondii in China may be highly clonal with atypical genotypes and higher virulence.

Seroprevalence of Toxoplasma gondii infection in stray and household cats in Guangzhou, China.

The prevalence of anti-Toxoplasma gondii specific IgG in stray and household cats in Guangzhou, China was determined by ELISA on serum samples from 206 cats (117 strays and 89 households) and the overall infection rate was 25.24%. The infection rate in stray cats (30.77%) was significantly higher (P < 0.05) than in household cats (17.98%). The rate of infection between male and female cats of both groups was not significantly different (P > or = 0.05), 28.13% versus 32.61% for male and female in stray cats, respectively, and 18% versus 17.95% in household cats. The present investigation demonstrated that the prevalence of T. gondii infection in cats in Guangzhou was high, especially in stray cats, which are probably the main source of T. gondii infection in this area. Integrated control strategies and measures should be implemented to prevent and control T. gondii infection in both stray and household cats, which will have significant implications for the control of human infection with T. gondii.

Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential.

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5-1 ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.

The presence of Mycoplasma hominis in isolates of Trichomonas vaginalis impacts significantly on DNA fingerprinting results.

The genetic characterization of Trichomonas vaginalis (Protista: Trichomonadidae), the causative agent of trichomoniasis in humans, is central to understanding the epidemiology, treatment, drug resistance, and virulence as well as the diagnosis and control of this parasite. Various molecular approaches, including DNA fingerprinting, have been employed for this purpose, and random amplification of polymorphic DNA (RAPD) continues to be utilized. However, little attention has been paid to the fact that some T. vaginalis populations can harbor symbiotic Mycoplasma hominis and/or other agents, which could cause artifacts in the RAPD results. In the present study, we demonstrate clearly that the presence of M. hominis from T. vaginalis isolates impacts significantly on RAPD results and on the subsequent analyses and interpretation of data sets. Moreover, symbiotic M. hominis displays an isolate-to-isolate variability in RAPD profile before elimination, suggesting a variability of M. hominis infection.

Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice.

The beta-tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta-tubulin shows 100%, 99.8%, 99.1%, and 98.6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51.6% of homology. Recombinant beta-tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta-tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83.3%, 70% and 76.7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta-tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta-tubulin recognized only T. evansi beta-tubulin and not mouse beta-tubulin. The results of this study demonstrated that the recombinant T. evansi beta-tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

Symbiosis of Mycoplasma hominis in Trichomonas vaginalis may link metronidazole resistance in vitro.

Fourteen of 28 Trichomonas vaginalis isolates collected from patients in Guangzhou, China from 2003 to 2004 were found to be naturally infected with Mycoplasma hominis, as determined by PCR using specific primers. In vitro metronidazole sensitivity assay of the 28 isolates revealed four displaying low susceptibility [minimum lethal concentration (MLC)= approximately 13-25 microg/ml] and another four displaying high resistance (MLC=50-100 microg/ml). The overwhelming majority of these resistant isolates (7/8) were mycoplasma-infected. The mean of MLCs of mycoplasma-infected isolates is approximately 10-fold higher than the mean of noninfected isolates (p=0.029). Sequence analyses of PCR-amplified small subunit-large subunit rRNA interspacer regions (ITS1/5.8S/ITS2) revealed that 23 of the 28 samples are identical, the remaining five being separable into two groups, each with a single point mutation. These internal transcribed spacer sequence variants are associated neither with mycoplasma infection nor with drug resistance. In contrast, random amplified polymorphic DNA analyses of DNAs using 10 different primers showed that the drug-resistant isolates are clustered together in association with mycoplasma infection, albeit more loosely. Taken together, the results obtained from this study suggest that in vitro metronidazole resistance of T. vaginalis is related to mycoplasma infection of this protozoan.

Survey of helminths in adult sheep in Heilongjiang Province, People's Republic of China.

The prevalence of helminths in adult sheep was investigated in Heilongjiang Province, People's Republic of China between January 1999 and September 2003. A total of 326 adult sheep representing local breeds (Xingjiang Fine Wool Sheep, Dongbei Fine Wool Sheep) as well as introduced breeds (Merino and Charollais) from representative geographical locations in Heilongjiang Province were slaughtered and examined for the presence of helminths. The worms were examined, counted and identified to species according to existing keys and descriptions. A total of 26 helminth species were found representing 2 phyla, 3 classes, 13 families and 20 genera. All sheep were infected by more than one helminth species. Oesophagostomum columbianum, Haemonchus contortus and Trichostrongylus colubriformis were the most common nematode species, and Paramphistomum cervi, Orientobilharzia turkestanica and Fasciola hepatica were the most common trematode species, whereas the infection of adult sheep with cestodes was uncommon. The results of the present investigation provide relevant "base-line" data for Heilongjiang Province, China, for assessing the effectiveness of future control strategies against helminth infections in sheep.

Application of a PCR assay to enhance the detection and identification of Tritrichomonas foetus in cultured preputial samples.

The traditional diagnostic test for Tritrichomonas foetus involves collection of preputial or vaginal samples followed by culture in a growth media and microscopic examination. Recently, polymerase chain reaction (PCR) techniques have been described for use as a diagnostic assay. The objective of this study was to evaluate a previously described PCR assay for detecting T. foetus in cultured preputial material. The detection limits of the assay for T. foetus organisms in a growth medium, in samples prepared from washing microscope slides, and in preputial material cultured in a growth medium were determined. Preputial samples were collected from 13 bulls uninfected with T. foetus. The PCR assay was able to detect 5 T. foetus organisms in the growth medium and the cultured preputial material. Amplification products were obtained from samples prepared from washes of microscope slides containing as few as 3 visualized organisms. The PCR assay was able to detect organisms in culture at a lower concentration than was possible by direct microscopic examination. This low detection limit may allow the PCR assay to be used to enhance the sensitivity of the current diagnostic test. In addition, the assay could be used to confirm the identification of T. foetus organisms observed by direct microscopic examination when other confirmation techniques, such as staining and phase microscopy, are not practical.

High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli.

Surface antigen 1 (SAG1) of Toxoplasma gondii is a good candidate for diagnosis and vaccine development, but recombinant SAG1 produced in Escheichia coli often loses its specific immunogenicity due to the incorrect folding. In the present study, a truncated SAG1 was highly expressed in E. coli as a fusion protein, about 30% of the total protein of the cell lysate. After a simple purification and refolding procedure, purified rSAG1 can be recognized by human Toxoplasma-infective serum, and ELISA kits constructed by rSAG1 can sensitively and specifically detect toxoplasma infection.

Analysis of the CD40/CD40L role in the sustenance of alloreactive antibody production.

CD40 ligand (CD40L) is important for T/B lymphocyte interaction. To understand the cellular basis of humoral allosensitization we, therefore: (1) measured CD40L protein and gene expression in sensitized and non-sensitized uremic unactivated peripheral CD4+ T lymphocytes; (2) studied the impact of blocking the CD40/CD40L pathway on alloreactive antibody (allo-Ab) production by engrafted sensitized PBLs into severe combined immunodeficient (SCID) mice after in vitro preactivation with IL2/LPs/HLA class II allopeptides and adjuvants as a potent stimulus to produce allo-Ab (Shoker et al. Transplantation 1999;68;1188); and (3) studied the modifying effect of CD40/CD40L blockade on T helper type I and II cytokine gene expression in the respective mice spleen. The CD40L protein was measured by flow cytometry and the gene expression was measured by quantitative RT-PCR. Alloreactive antibodies (alo-Abs) produced by sensitized PBLs engrafted into SCID mice with and without blockade of the CD40 receptor were measured by the PRA-STAT ELISA method. The modifying effects of CD40 blocking on allo-Ab production and cytokine gene expression by the engrafted cells measured by RT-PCR were then compared. There was no detectable CD40L protein expression in either the uremic or the control groups. The CD40L gene expression of 0.04 +/- 0.02 attomoles (aM) in the sensitized group was significantly higher than in the non-sensitized patients (0.009 +/- 0.007 aM, P < 0.0001) or the control CD4+ T cells (0.016 +/- 0.004 aM, P < 0.001). Blockade of the CD40 receptor abrogated the production of allo-Ab antibodies by the engrafted sensitized cells in 60% of the tested mice (n = 10); decreased the mean +/- S.D. optic density of allo-Ab to 0.1 +/- 0.13 and the mean +/- S.D. PRA to 12 + 16). In the presence of the control Ab, allo-Ab production in SCID sera was present in 100% of the 10 SCID mice tested; the mean +/- S.D. PRA was 75 +/- 20, and the mean + S.D. OD activity was 0.412 +/- 0.17. All cytokine genes were, otherwise, expressed in the presence or absence of CD40 blockade. The results suggest a potential role of an enhanced CD40/CD40L interaction in the sustenance of alloreactive antibody production without significant deviation to T helper-like I or II responses. Blocking the CD40/CD40L pathway may have a potential therapeutic benefit to treat sensitized uremic patients.

Trypanosoma evansi and T. equiperdum: distribution, biology, treatment and phylogenetic relationship (a review).

Trypanosoma evansi and T. equiperdum were compared regarding their ultrastructure, their mammalian hosts, way of transmission, pathogenicity, diagnosis and treatment, and biochemical and molecular characteristics. Electron microscopic investigation revealed no ultrastructural differences between the two species except that there were more coated vesicles in the flagellar pocket of T. equiperdum. Biological, biochemical and molecular studies were reviewed and exhibited many similarities between T. evansi and T. equiperdum. The most prominent differences between the two species are the presence of maxicircles in T. equiperdum, which are missing in T. evansi, and the route of transmission. While T. evansi is transmitted by biting flies, T. equiperdum is transmitted from one equine host to another during copulation when mucous membranes come into contact. Otherwise the two species are remarkably similar. The phylogenetic relationship between the two species and T. b. brucei is being discussed, and the hypothesis is proposed that T. evansi arose from a clone of T. equiperdum which lost its maxicircles.

Chromosome numbers of Tritrichomonas foetus and Tritrichomonas suis.

Tritrichomonas foetus and Tritrichomonas suis isolates were cultivated axenically in Diamond's medium. Studies on the chromosome numbers of these two species with a light microscope were done by adding different concentrations of colchicine into the medium, incubating at 37 degrees C for 6-8 h and using a hypotonic swelling technique. The diploid chromosome numbers of both T. foetus and T. suis were 2n=10.

Susceptibility of dyskinetoplastic Trypanosoma evansi and T. equiperdum to isometamidium chloride.

Isometamidium chloride (ISM) is an effective trypanocide with curative and prophylactive activity in husbandry animals. The mode of action of ISM against pathogenic trypanosomes is not fully understood, but there is evidence in the literature that kinetoplastic topoisomerase type II is selectively inhibited by the drug. This prompted the hypothesis that dyskinetoplastic trypanosomes would express a reduced level of susceptibility to ISM. From parental Trypanosoma evansi and T. equiperdum populations we generated clones containing only dyskinetoplastic organisms and clones containing organisms with kinetoplasts. The susceptibility of these clones to ISM was quantified by in vitro assays. The susceptibility of all clones was in the same range. Minor differences in drug susceptibility found between the clones showed that the dyskinetoplastic T. evansi and T. equiperdum clones were even more susceptible to ISM than their kinetoplastic counterparts. Thus, the hypothesis that the dyskinetoplastic trypanosomes would be less susceptible to or tolerant of ISM was clearly disproved. The previously demonstrated inhibition of kinetoplastic topoisomerase type II by ISM cannot be the primary toxic effect of the drug on trypanosomes, and the mode of action of ISM needs to be reassessed.