Characterization of a spontaneous cyst-forming strain of Toxoplasma gondii isolated from Tokachi subprefecture in Japan.

Apicomplexan parasite Toxoplasma gondii has three distinct clonal lineages: high, medium and low virulent strains, type I, II and III, respectively. T. gondii avoids the immune response by transforming from fast multiplying tachyzoite to slow multiplying bradyzoite, and establishing a chronic infection. In the present study, we isolated a new strain of T. gondii from cat feces in the Tokachi subprefecture, Hokkaido, Japan and named it as TgCatJpObi1 (Obi1) strain. Genotyping analysis of 12 loci revealed atypical characters close to type II, genotype 4 according to ToxoDB classification. Phenotypically, Obi1 strain shows slow growth rate and the ability of spontaneous cyst formation in both human foreskin fibroblast (HFFs) and mouse peritoneal macrophages in vitro without bradyzoite induction. Parasite virulence was assessed by means of mouse survival upon infection with either Obi1 or ME49 strains. Obi1 strain displayed no mortalities in comparison to type II clonal lineage, ME49 at LD50 to LD100 range (1 × 103-106 tachyzoites). Although virulence of Obi1 strain is significantly lower than that of ME49, nucleotide sequences analyses revealed that genes of virulence factors such as Gra15, Rop5, 16, 17, and 18 in Obi1 strain were 100% identical to those in the type II strain. Thus, characterization of a newly isolated strain, Obi1, is crucial to clarify the development of toxoplasmosis in both humans and animals.

Genetic analysis of Cryptozona siamensis (Stylommatophora, Ariophantidae) populations in Thailand using the mitochondrial 16S rRNA and COI sequences.

Cryptozona siamensis, one of the most widespread land snails, is native to Thailand, and plays a key role as an agricultural pest and intermediate host for Angiostrongylus spp. However, its genetic diversity and population structure has not yet been investigated, and are poorly understood. Therefore, a genetic analysis of the C. siamensis population in Thailand was conducted, based mitochondrial 16S rRNA (402 bp) and COI (602 bp) gene fragment sequences. Cryptozona siamensis randomly collected from 17 locations in four populations across Thailand, between May 2017 and July 2018. Fifty-eight snails were used to examine the phylogeny, genetic diversity, and genetic structure. The maximum likelihood tree based on the 16S rRNA and COI fragment sequences revealed two main clades. A total of 14 haplotypes with 44 nucleotide variable sites were found in the 16S rRNA sequences, while 14 haplotypes with 57 nucleotide variable sites were found in the COI sequences. The genetic diversity of C. siamensis in term of the number of haplotypes and haplotype diversity, was found to be high but the nucleotide diversity showed low levels of genetic differentiation for the COI sequence as also noted with the 16S rRNA sequence. The population genetic structure of C. siamensis revealed genetic difference in most populations in Thailand. However, low genetic difference in some populations may be due to high gene flow. This study provides novel insights into the basic molecular genetics of C. siamensis.

Prevalence of Toxoplasma gondii and other intestinal parasites in cats in Tokachi sub-prefecture, Japan.

The present study investigated the prevalence of Toxoplasma gondii and other intestinal parasites in cats in the Tokachi subprefecture in Japan. A total of 365 household cats were included in the study, and 353 serum and 351 fecal samples were collected and analyzed. T. gondii IgG antibodies were detected in the sera of 16.14% of cats based on Latex agglutination test and ELISA. For ELISA, T. gondii RH strain tachyzoites lysate and T. gondii SAG2 recombinant protein were used as antigens. Low seropositivity was detected in cats younger than one year and older than 11 years; outdoor and hunter cats showed significantly high seropositivities. Neutering either in male or female cats, but not gender, had a considerable effect on seroprevalence. Toxoplasma gondii oocysts were detected in one fecal sample. The overall parasitic infestation in cats was 12.5%. Other detected parasites included Toxocara species, which showed the highest prevalence of 7.7%, followed by Isospora spp. (2%), Taenia spp. (1.7%), and Ancylostoma spp. (0.9%). Spirometra spp. was detected in only one sample. Outdoor cats comprised 50% of all 44 parasite-infested cats. Although T. gondii oocysts were detected in only one sample, the relatively high seroprevalence of T. gondii indicated that it can pose significant risks to the environment. Our findings highlighted the potential of outdoor cats as a source of T. gondii and other parasites.

Potential risk of a liver fluke Opisthorchis viverrini infection brought by immigrants from prevalent areas: A case study in the lower Northern Thailand.

Considering the long lifespan of the liver fluke Opisthorchis viverrini, human mobility from prevalent regions to other neighboring areas has the possibility to disperse carriers and complicate the opisthorchiasis problem. To evaluate this, mass screening of the fluke infection was conducted in nine communities of lower Northern Thailand, combined with a questionnaire survey to distinguish the participant's origin. The liver fluke infection was found in 70 individuals (7.2%) of the examined 971 stool samples from seven communities, with light intensity providing small numbers of eggs in the examined stool. Prevalence in the positive communities varied from 2.1% to 28.7%. As a result of generalized linear mixed models fitting, regional origin and raw-fish eating habits were stably selected as variables affecting the parasite infection while occupation and educational background were secondary ones. Majority of the infected cases (64.3%) were found from the immigrants of northeastern Thailand (the fluke prevalent region), providing 2.28-2.42 times higher infectious risk on average against the local residents. Daily consumption of raw fish averaged a 3.12-3.60 times higher risk compared to those with no raw-fish eating habit. Our findings suggest that people's origin and moving history deserve further attentions in health promotion programs including education for safe eating.

Prevalence of gastrointestinal helminth parasites of zoonotic significance in dogs and cats in lower Northern Thailand.

Gastrointestinal zoonotic helminths of dogs and cats have a public health concern worldwide. We investigated the prevalence of gastrointestinal helminths of zoonotic significance in dogs and cats in lower Northern Thailand and utilized molecular tools for species identification of hookworms and Opisthorchis viverrini. Fecal samples of 197 dogs and 180 cats were collected. Overall prevalence of infection using microscopy was 40.1% in dogs and 33.9% in cats. Helminth infection found in both dogs and cats included hookworms, Spirometra spp., Taenia spp., Toxocara spp., O. viverrini, Strongyloides spp. and Trichuris spp. Hookworms were the most common helminth in dogs, while Spirometra spp. were the most prevalent in cats. Among hookworm infection in dogs and cats, Ancylostoma ceylanicum was the most prevalent hookworm, being 82.1% in hookworm infected dogs and 95.8% in hookworm infected cats. Mixed-infection due to hookworms and Spirometra spp. was the most dominant in both dogs and cats. Our finding showed that zoonotic helminth infection is highly prevalent in dogs and cats in the lower Northern area of Thailand.

Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.

Binding to complement factors and activation of the alternative pathway by Acanthamoeba.

Acanthamoeba can cause severe ocular and cerebral diseases in healthy and immunocompromised individuals, respectively. Activation of complement appears to play an important role in host defence against infection. The exact mechanism, however, is still unclear. The aim of the present study was to investigate the effect of normal human serum (NHS) and normal mouse serum (NMS) on Acanthamoeba trophozoites, the binding of different complement factors to Acanthamoeba and the activation of the complement system. Moreover, we aimed to work out any possible differences between different strains of Acanthamoeba. A virulent T4 strain, a non-virulent T4 strain and a virulent T6 strain were included in the study. It was shown that NHS, but not NMS clearly has amoebicidal properties. After 5min of incubation with NHS, amoebae showed plasma membrane disruption and extrusion of intracellular components. Cells were completely destroyed within 60min of incubation in NHS but stayed intact after incubation in heat-inactivated serum. The binding of human C3 and C9 to amoebae was established by immunoblotting. Although incubation with mouse serum did not result in lysis of Acanthamoeba trophozoites an immunofluorescence assay (IFA) demonstrated a strong deposition of mouse complement factor C3 activation products, moderate binding of C1q, but no binding of MBL-A and MBL-C. EDTA inhibited the binding of C3 to acanthamoebae. Binding of amoebae to C3b was observed with sera from C1qa-/- and MBL-A/C-/- mice, but not with serum from Bf/C2-/- mice demonstrating an activation of complement via the alternative pathway. There were no significant differences between the three Acanthamoeba strains investigated. Altogether, our results prove that NHS is amoebolytic and that Acanthamoeba binds to C3 and C9 and activates the complement system via the alternative pathway.

Acanthamoeba strains show reduced temperature tolerance after long-term axenic culture.

Acanthamoeba is a genus of free-living organisms that can be found in various habitats. We investigated the physiological characteristics of 15 Acanthamoeba isolates, representing five genotypes (T4, T5, T6, T7, and T11) of both clinical and nonclinical origins. Moreover, in order to evaluate possible alterations from long-term culture, old and fresh isolates were included, and results were compared to a previous study. We found that there is no significant difference in physiological characteristics between genotypes. However, Acanthamoeba strains that had been grown in axenic culture over long periods of time adapted to axenic growth. Overall growth rates under-agarose migration and particularly, temperature tolerance decrease after long-term axenic culture at room temperature. The only trait that remained rather constant was the cytopathic effect.

Detection of Paragonimus heterotremus eggs in experimentally infected cats by a polymerase chain reaction-based method.

A polymerase chain reaction (PCR) procedure for the detection of Paragonimus heterotremus eggs in stool samples was developed and compared with Stoll's egg count method. The primers were designed on the basis of a previously constructed pPH-13-specific DNA probe, which produced an approximate 0.5-kb amplified product. This PCR method could detect as few as 5 eggs in 0.6 g of artificially inoculated feces of a healthy control cat or as little as 1 x 10(-4) ng of P. heterotremus genomic DNA. The assay had 100% sensitivity in all infected cats. The method did not yield an approximate 0.5-kb product with DNA from other parasites such as Gnathostoma spinigerum, Trichinella spiralis, Fasciola gigantica, Echinostoma malayanum, Opisthorchis viverrini, Dirofilaria immitis, and Taenia saginata; exceptions were Paragonimus siamensis and Paragonimus westermani. In addition, no genomic DNA from Escherichia coli, Burkholderia pseudomallei, Acinetobacter anitratus, Mycobacterium tuberculosis, Staphylococcus aureus, beta-Streptococcus grA, and Proteus mirabilis or from the vertebrate and invertebrate hosts of P. heterotremus was amplified in the PCR assay. This assay has great potential for application in clinical epidemiological studies.

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood.

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.

Culex quinquefasciatus in Phitsanulok as a possible vector of nocturnally periodic Wuchereria bancrofti transmission in Myanmar immigrants.

The present study was undertaken in order to study whether Culex quinquefasciatus collected in Phitsanulok Province can be an insect host for the development of Wuchereria bancrofti larvae. W. bancrofti infected blood from Myanmar workers in Mae Sot, Tak Province was fed to mosquitoes by using the artificial membrane feeding. An infection of W. bancrofti was found with the highest density of L3 in the mosquito thorax on the 14th day after feeding. The infection rate also correlated to the density of microfilaria found in the donor's blood. Our results showed that Cx. quinquefasciatus in Phitsanulok is a possible vector of nocturnally periodic W. bancrofti.