Naegleria fowleri causes a deadly infection known as primary amoebic meningoencephalitis (PAM). To our knowledge, there are very few transcriptome studies conducted on these brain-eating amoebae, despite rise in the number of cases. Although the Naegleria genome has been sequenced, currently, it is not well annotated. Transcriptome level studies are needed to help understand the pathology and biology of this fatal parasitic infection. Recently, we showed that nanoparticles loaded with the flavonoid Hesperidin (HDN) are potential novel antimicrobial agents. N. fowleri trophozoites were treated with and without HDN-conjugated with silver nanoparticles (AgNPs) and silver only, and then, 50% minimum inhibitory concentration (MIC) was determined. The results revealed that the MIC of HDN-conjugated AgNPs was 12.5 microg/mL when treated for 3 h. As no reference genome exists for N. fowleri, de novo RNA transcriptome analysis using RNA-Seq and differential gene expression analysis was performed using the Trinity software. Analysis revealed that more than 2000 genes were differentially expressed in response to N. fowleri treatment with HDN-conjugated AgNPs. Some of the genes were linked to oxidative stress response, DNA repair, cell division, cell signalling and protein synthesis. The downregulated genes were linked with processes such as protein modification, synthesis of aromatic amino acids, when compared with untreated N. fowleri. Further transcriptome studies will lead to understanding of genetic mechanisms of the biology and pathogenesis and/or the identification of much needed drug candidates.
Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 μg/mL. The IC50 was found to be between 100 and 50 μg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.
Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.
Enterocytozoon bieneusi is an emerging opportunistic pathogen infecting humans, and both domestic and wild pigs are known to harbour zoonotic genotypes. There remains a paucity of information on the prevalence and epidemiology of this enteropathogen in Southeast Asia. The present study was undertaken to determine the molecular prevalence and risk factors associated with E. bieneusi infection among commercially farmed pigs in Malaysia. Faecal samples were collected from 450 pigs from 15 different farms and subjected to nested PCR amplification of the ribosomal internal transcribed spacer (ITS) gene of E. bieneusi. Phylogenetic analysis involved 28 nucleotide sequences of the ITS region of E. bieneusi. An interviewer-administered questionnaire provided information on the animal hosts, farm management systems and environmental factors and was statistically analysed to determine the risk factors for infection. The prevalence of E. bieneusi infection was relatively high (40.7%). The highest prevalence (51.3%) was recorded among the piglets, while the adults showed the lowest level of infection (31.3%). Multivariate analysis indicated that age of the pigs, distance of the farm from human settlement and farm management system were significant risk factors of infection. Three genotypes (EbpA, EbpC and Henan-III) detected among the pigs are potentially zoonotic. The high prevalence of E. bieneusi among locally reared pigs, the presence of zoonotic genotypes and the spatial distribution of pig farms and human settlements warrant further investigation on the possibility of zoonotic transmission.
To date, more than 50 Eimeria spp. have been isolated from marsupials of the family Macropodidae. Although 18 species of Eimeria have been previously detected from multiple animal species belonging to the genus Macropus of the family, limited genetic analyses of the parasites are available, and their pathogenicity remains unclear. Here, we report the isolation of Eimeria spp. from a zoo specimen of red-necked wallaby (Macropodidae; Macropus rufogriseus). Specifically, two distinct types of Eimeria oocysts were recovered, one from the feces before treatment with an anthelmintic and the second from the intestinal contents after death of the animal. The oocysts obtained from the two sources were morphologically identified as E. hestermani and E. prionotemni, respectively. We successfully determined partial gene sequences from the two isolates, including segments of the 18S rRNA genes, and for the first time have used phylogenetic analyses of these sequences to assign the species to distinct clades. In combination with further genetic data, these results are expected to help elucidate the pathogenicity and host ranges of Eimeria spp. within the respective family and genus.
Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.
Examination of 35 barramundi (Lates calcarifer) from aquaculture cages in Setiu Wetland, Malaysia, revealed a single fish infected with three Henneguya spp. (Cnidaria: Myxosporea). Characterization of the infections using tissue tropism, myxospore morphology and morphometry and 18S rDNA sequencing supported description of three new species: Henneguya setiuensis n. sp., Henneguya voronini n. sp. and H. calcarifer n. sp. Myxospores of all three species had typical Henneguya morphology, with two polar capsules in the plane of the suture, an oval spore body, smooth valve cell surfaces, and two caudal appendages. Spores were morphometrically similar, and many dimensions overlapped, but H. voronini n. sp. had shorter caudal appendages compared with H. calcarifer n. sp. and H. setiuensis n. sp. Gross tissue tropism distinguished the muscle parasite H. calcarifer n. sp. from gill parasites H. setiuensis n. sp. and H. voronini n. sp.; and these latter two species were further separable by fine-scale location of developing plasmodia, which were intra-lamellar for H. setiuensis n. sp. and basal to the filaments for H. voronini n. sp. small subunit ribosomal DNA sequences distinguished all three species: the two gill species H. setiuensis n. sp. and H voronini n. sp. were only 88% similar (over 1708 bp), whereas the muscle species H. calcarifer n. sp. was most similar to H. voronini n. sp. (98% over 1696 bp). None of the three novel species was more than 90% similar to any known myxosporean sequence in GenBank. Low infection prevalence of these myxosporeans and lack of obvious tissue pathology from developing plasmodia suggested none of these parasites are currently a problem for barramundi culture in Setiu Wetland; however additional surveys of fish, particularly at different times of the year, would be informative for better risk assessment.
Chrysomya bezziana is an obligate, myiasis-causing fly in humans and warm-blooded animals throughout the tropical and subtropical Old World. We report a case of cutaneous myiasis due to C. bezziana in a dog from Guangxi province in China. A total of 35 maggots were removed from the lesions. Direct sequencing of the mitochondrial cytochrome b gene showed that the specimen belonged to haplotype CB_bezz02, which was previously reported in Malaysia and the Gulf region. This paper also reviews reported cases of screwworm myiasis from humans and animals in China. Geographical records indicate that the distribution of C. bezziana is expanding, suggesting that an integrated pest management control should be taken into consideration in China.
The geographical distribution of tuberculosis (TB) overlaps with various parasitic infections. Uncovering the characteristics of coinfecting parasites that potentially affect the host susceptibility to TB is pertinent as it may provide input to current TB therapeutic and prophylactic measures. The present study was aimed at examining the types of parasitic infections in TB patients and healthy TB contacts (HC) in Orang Asli, Malaysian aborigines, who dwelled in the co-endemic areas. Stool and serum samples were collected from Orang Asli who fulfilled the selection criteria and provided written informed consents. Selected parasitic infections in the two study groups were determined by stool examination and commercial serum antibody immunoassays. The prevalence of parasitic infections in TB and HC participants were 100% (n = 82) and 94.6% (n = 55) respectively. The parasitic infections comprised toxocariasis, trichuriasis, amoebiasis, toxoplasmosis, hookworm infection, ascariasis, strongyloidiasis, and brugian filariasis, in decreasing order of prevalence. Overall, helminth or protozoa infection did not show any significant association with the study groups. However, when the species of the parasite was considered, individuals exposed to trichuriasis and toxoplasmosis showed significant odds reduction (odds ratio (OR) 0.338; 95% confidence interval (CI) 0.166, 0.688) and odds increment (OR 2.193; 95% CI 1.051, 4.576) to have active pulmonary TB, respectively. In conclusion, trichuriasis and toxoplasmosis may have distinct negative and positive associations respectively with the increase of host susceptibility to TB.
Simulium dermatitis is an IgE-mediated skin reaction in animals and humans caused by the bites of black flies. Although Simulium nigrogilvum has been incriminated as the main human-biting black fly species in Thailand, information on its salivary allergens is lacking. Salivary gland extract of S. nigrogilvum females was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the separated components were applied onto nitrocellulose membranes for immunoblotting, which was performed by probing the protein blots with sera from 17 individuals who were allergic to the bites of S. nigrogilvum. IgE-reactive protein bands were characterized further by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Nine protein bands (79, 42, 32, 25, 24, 22, 15, 13, and 11 kDa) were recognized in the serum of the subjects. Four of the nine protein bands (32, 24, 15, and 11 kDa) showed IgE reactivity in all (100%) of the tested sera, and they were identified as salivary secreted antigen 5-related protein, salivary serine protease, erythema protein, and hypothetical secreted protein, respectively. Three other proteins, salivary serine protease (25 kDa), salivary D7 secreted protein (22 kDa), and hypothetical protein (13 kDa), reacted with > 50% of the sera. The relevance of the identified protein bands as allergens needs to be confirmed by using pure recombinant proteins, either in the in vivo skin prick test or in vitro detection of the specific IgE in the serum samples of allergic subjects. This will be useful for the rational design of component-resolved diagnosis and allergen immunotherapy for the allergy mediated by the bites of black flies.
Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid-conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid-AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5 μM concentration of oleic acid alone and oleic acid-conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid-conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5 μM. Furthermore, oleic acid and oleic acid-conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid-conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.
A morphological and molecular phylogenetic study of proteocephalid tapeworms of the genus Acanthotaenia von Linstow, 1903, parasites of monitors (Varanidae), was carried out. The type species, A. shipleyi von Linstow, 1903, which was originally described based on an immature specimen from Sri Lanka, is redescribed based on new material from the type host, Varanus salvator, in Sri Lanka, Malaysia, and Vietnam, and its neotype is designated. In addition, Acanthotaenia susanae n. sp. is described from Varanus nebulosus in Vietnam. The new species differs from congeners by the large size of the scolex, width of the rostellum and the number of testes. New molecular data (sequences of lsrDNA and cox1) revealed Acanthotaenia paraphyletic with the inclusion of Australotaenia bunthangi de Chambrier & Scholz, 2012, a parasite of Enhydris enhydris (Ophidia: Homalopsidae) in Cambodia. Molecular data confirm a wide distribution of A. shipleyi (isolates from Malaysia and Vietnam were almost identical) and indicate a strict host specificity (oioxeny) of individual species of the genus. Type specimens of four species made it possible to supplement their morphological descriptions. A survey of all species of Acanthotaenia recognised as valid is presented and the following taxonomic changes are proposed: Acanthotaenia pythonis Wahid, 1968 described from the green python, Morelia viridis, in a zoo, is transferred to Kapsulotaenia as Kapsulotaenia pythonis (Wahid, 1968) n. comb., because it possesses intrauterine eggs grouped in capsules. Acanthotaenia gracilis (Beddard, 1913) from Varanus varius in Australia is considered to be species inquirenda because its original descriptions did not contain sufficient data for adequate circumscription and differentiation from congeners and type material was not available. Generic diagnosis of Acanthotaenia is amended and a key to its seven species is provided.
Eliminating the Plasmodium vivax malaria parasite infection remains challenging. One of the main problems is its capacity to form hypnozoites that potentially lead to recurrent infections. At present, primaquine is the only drug used for the management of hypnozoites. However, the effects of primaquine may differ from one individual to another. The aim of this work is to determine new measures to reduce P. vivax recurrence, through primaquine metabolism and host genetics. A genetic study of MAO-A, CYP2D6, CYP1A2 and CYP2C19 and their roles in primaquine metabolism was undertaken of healthy volunteers (n = 53). The elimination rate constant (Ke) and the metabolite-to-parent drug concentration ratio (Cm/Cp) were obtained to assess primaquine metabolism. Allelic and genotypic analysis showed that polymorphisms MAO-A (rs6323, 891G>T), CYP2D6 (rs1065852, 100C>T) and CYP2C19 (rs4244285, 19154G>A) significantly influenced primaquine metabolism. CYP1A2 (rs762551, -163C>A) did not influence primaquine metabolism. In haplotypic analysis, significant differences in Ke (p = 0.00) and Cm/Cp (p = 0.05) were observed between individuals with polymorphisms, GG-MAO-A (891G>T), CT-CYP2D6 (100C>T) and GG-CYP2C19 (19154G>A), and individuals with polymorphisms, TT-MAO-A (891G>T), TT-CYP2D6 (100C>T) and AA-CYP2C19 (19154G>A), as well as polymorphisms, GG-MAO-A (891G>T), TT-CYP2D6 (100C>T) and GA-CYP2C19 (19154G>A). Thus, individuals with CYP2D6 polymorphisms had slower primaquine metabolism activity. The potential significance of genetic roles in primaquine metabolism and exploration of these might help to further optimise the management of P. vivax infection.
The Simulium rufibasis subgroup is one of three subgroups of the Simulium (Simulium) tuberosum species-group; it is characterized by a pair of clustered stout hairs on the ventral surface of female abdominal segment 7. A member of the S. rufibasis subgroup in Taiwan was investigated morphologically and genetically using the universal cytochrome c oxidase subunit I (COI) barcoding gene and polytene chromosomal banding pattern. The Taiwanese material is morphologically similar to S. rosliramlii Takaoka & Chen from Vietnam and represents the second species of the S. rufibasis subgroup known from Taiwan. It also represents a novel molecular lineage that is distinct from three other primary lineages identified as S. doipuiense, S. doipuiense/S. rufibasis, and S. weji previously reported from Thailand. The mitochondrial evidence for a distinct lineage in Taiwan is supported by chromosomal analysis, which revealed unique sex chromosomes. For nomenclatural stability, we associate the name S. arisanum Shiraki with the Taiwanese entity. Originally described from females from Taiwan, S. arisanum until now has remained an enigmatic species.
Blastocystis sp. is a gastrointestinal (GI) protozoan parasite reported to cause non-specific GI symptoms including diarrhea, flatulence, abdominal pain, and nausea. Complete eradication of Blastocystis sp. is rather challenging even with the drug of choice, i.e., metronidazole. Here, we report on two Blastocystis sp.-infected individuals, who presented increased parasite load and exacerbated symptoms upon treatment with the usual recommended dosage and regime of metronidazole. The two studies uniquely demonstrate for the first time a cyst count as high as fivefold more than the original cyst count before treatment and show an exacerbation of GI symptoms despite treatment. The study provides additional support in recognizing metronidazole resistance in Blastocystis sp. and its consequences towards the pathogenicity of the parasite.
Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.
This work investigated the anti-amoebic activity of two samarium (Sm) complexes, the acyclic complex [bis(picrato)(pentaethylene glycol)samarium(III)] picrate-referred to as [Sm(Pic)2(EO5)](Pic)-and the cyclic complex [bis(picrato)(18-crown-6)samarium(III)] picrate-referred to as [Sm(Pic)2(18C6)](Pic). Both Sm complexes caused morphological transformation of the protozoa Acanthamoeba from its native trophozoite form carrying a spine-like structure called acanthopodia, to round-shaped cells with loss of the acanthopodia structure, a trademark response to environmental stress. Further investigation, however, revealed that the two forms of the Sm complexes exerted unique cytotoxicity characteristics. Firstly, the IC50 of the acyclic complex (0.7 μg/mL) was ~ 10-fold lower than IC50 of the cyclic Sm complex (6.5 μg/mL). Secondly, treatment of the Acanthamoeba with the acyclic complex caused apoptosis of the treated cells, while the treatment with the cyclic complex caused necrosis evident by the leakage of the cell membrane. Both treatments induced DNA damage in Acanthamoeba. Finally, a molecular docking simulation revealed the potential capability of the acyclic complex to form hydrogen bonds with profilin-a membrane protein present in eukaryotes, including Acanthamoeba, that plays important roles in the formation and degradation of actin cytoskeleton. Not found for the cyclic complex, such potential interactions could be the underlying reason, at least in part, for the much higher cytotoxicity of the acyclic complex and also possibly, for the observed differences in the cytotoxicity traits. Nonetheless, with IC50 values of
The ornamental fish trade provides a pathway for the global translocation of aquatic parasites. We examined a total of 1020 fish imported from Singapore, Malaysia, Thailand, or Sri Lanka to Australia (including freshwater and marine fish species) for monogenean ectoparasites. Fish were received following veterinary certification that they showed no clinical signs of pests and diseases from the exporting country and visual inspection at Australian border control. Australian import conditions require mandatory treatment for goldfish with parasiticides (e.g. trichlorfon, formaldehyde, sodium chloride) for the presence of gill flukes (Dactylogyrus vastator Nybelin, 1924 and Dactylogyrus extensus Mueller and Van Cleave, 1932) prior to export. Over 950 individual parasites were detected in five imported fish species, representing 14 monogenean species. Seven Dactylogyrus spp. including D. vastator and three Gyrodactylus spp. infected goldfish, Carassius auratus Linnaeus, 1758, from Malaysia, Singapore, and Thailand. Dactylogyrus ostraviensis Řehulka, 1988, infected rosy barb, Pethia conchonius Hamilton, 1822, from Singapore, Sri Lanka, and Thailand while two Trianchoratus spp. infected three spot gourami, Trichopodus trichopterus Pallas, 1970 and pearl gourami Trichopodus leerii Bleeker, 1852, from Sri Lanka. Urocleidoides reticulatus Mizelle & Price, 1964, infected guppy, Poecilia reticulata Peters, 1859, from Sri Lanka. The discovery of D. vastator in goldfish, as well as 13 other monogenean species, shows that pre-export health requirements, which include chemical treatment of goldfish, and inspection of all ornamental fish species did not prevent infection by monogeneans. Inspection prior to exportation and at border control must account for the highly cryptic nature of monogenean parasites and consider alternatives to current pre-export conditions and visual inspection at border control.
Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.
Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.