Evaluation of the host specificity of Eimeria uekii and Eimeria raichoi for Japanese rock ptarmigans by oocyst transfer to taxonomically related birds.

Eimeria spp. are protozoan parasites that are commonly found in a broad range of vertebrate hosts. These parasites generally exhibit strict host specificity, but some Eimeria spp. can infect groups of closely related species such as species within a genus or family. Compared with Eimeria spp. that infect livestock, limited information is available about such infections in wild animals including data on host specificity, virulence, and prevalence. The Japanese rock ptarmigan, Lagopus muta japonica, is an endangered bird belonging to the family Phasianidae, order Galliformes, and inhabits only alpine areas of Japan. In conservation efforts for these birds, two Eimeria spp., E. uekii and E. raichoi, were frequently detected. Here, we examined cross-transmission of the parasites to other bird species to characterize their infectivity as well as the development of experimental bird models to contribute to conservation programs by the oocyst transfer. Consequently, among the examined eight bird species (chickens, Japanese pheasants, turkeys, chukar partridges, quails, helmeted guineafowls and ducks), only turkeys (family Phasianidae, order Galliformes) could be infected with E. raichoi. However, the number of oocysts per feces was relatively low, and few parasites in the intestinal mucosa could be found by histopathological analyses. These results might indicate that E. uekii and E. raichoi are highly adapted to Japanese rock ptarmigans that inhabit the alpine zone although further studies are anticipated.

Evaluation of Cryptosporidium parvum oocyst inactivation following exposure to ultraviolet light-emitting diodes by in vitro excystation and dye staining assays.

Cryptosporidium spp. are protozoan parasites that are transmitted via fecal-oral routes and can exhibit chemical resistance. Chlorine resistance makes it very difficult to eliminate parasites present in contaminated drinking water. While the efficacy of ultraviolet light-emitting diodes (UV-LEDs) against microorganisms has been reported, the efficacy of UV-LEDs against Cryptosporidium spp. has not been fully evaluated. Here, we assessed the efficacy of UV-LEDs with peak wavelengths of 268, 275, 284, and 289 nm against Cryptosporidium parvum at various exposure times, with a fixed exposure distance, using two in vitro methods. Consequently, the time required for 2 log10 inactivation through the excystation method by UV-LEDs of 268, 275, 284, and 289 nm was estimated as 115.5, 104.1, 37.4, and 30.7 min, respectively. The propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining assays estimated the inactivation time as 311.3, 275.2, 60.6, and 39.1 min, respectively. Our results showed that UV-LED irradiation at longer wavelengths produced higher inactivation activity against C. parvum, which corroborates our previously reported in vivo assay results, although further study is needed to clarify the mechanism.

Efficacy of ultraviolet light-emitting diodes (UV-LED) at four different peak wavelengths against Cryptosporidium parvum oocysts by inactivation assay using immunodeficient mice.

As an alternative to using ultraviolet (UV) lamps, which are made with mercury that is toxic to the environment and human health, UV light-emitting diodes (UV-LEDs) are expected to be effective for inactivating microorganisms in water. Although UV-LEDs have been reported to be effective against bacteria and viruses, the effectiveness of UV-LEDs against Cryptosporidium parasites has not been fully evaluated. As we report here, we have developed an in vivo quantitative inactivation assay for C. parvum oocysts using immunodeficient mice. Using the assay, we evaluated the effectiveness of treatment by UV lamp (254 nm) at approximately 1000 µJ/cm2 (for 3 s at a distance of 95 mm) compared to inactivation by commercially available UV-LEDs (with peak wavelengths of 268, 275, 284, and 289 nm). The shed patterns of oocysts after treatment with 284- and 289-nm wavelength UV-LEDs were significantly delayed compared to that after treatment with a UV lamp. These findings provide the first suggestion that UV-LEDs are effective against these parasites, as assessed using commercially available 350-mA UV-LEDs under conditions of fixed exposure distance and time.

Long-term administration of excess zinc impairs learning and memory in aged mice.

Zinc (Zn) is an essential element, but excess amounts are known to cause neurotoxic effects. The risk of excessive Zn intake is increased by supplementing food intake with dietary supplements. Ageing affects many cellular processes that predispose individuals to neurodegeneration. Indeed, the prevalence of senile dementia such as Alzheimer's disease, Parkinson's disease, and vascular-type dementia increases with age. As such, we investigated the effects of long-term exposure to excess Zn on learning and memory in aged mice. ICR-JCL female mice (aged 26 weeks) were administered 0, 200, or 500 ppm Zn as zinc chloride in drinking water for 30 weeks. After 30-week administration, aged female animals were subjected to Y-maze, novel object recognition, and step-through passive avoidance tests. Chronic exposure to Zn did not inhibit learning and memory in the Y-maze test, but dose-dependently inhibited learning and memory in novel object recognition and step-through passive avoidance tests. These results indicate the potential for chronic Zn exposure to dose-dependently inhibit both long-term and novel object recognition memory. Results of microarray analysis revealed significant changes in gene expression of transthyretin and many olfactory receptors in the hippocampus of Zn-treated mice.

Transitions in morphological forms and rapid development of the asexual schizonts of Eimeria tenella through serial passaging in chicks.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests.

Development and application of a rapid and sensitive genotyping method for pharmacogene variants using the single-stranded tag hybridization chromatographic printed-array strip (STH-PAS).

Genetic polymorphisms contribute to inter-individual variability in the metabolism of multiple clinical drugs, including warfarin, thiopurines, primaquine, and aminoglycosides. A rapid and sensitive clinical assessment of various genome biomarkers is, therefore, required to predict the individual responsiveness of each patient to these drugs. In this study, we developed a novel genotyping method for the detection of nine pharmacogene variants that are important in the prediction of drug efficiency and toxicity. This genotyping method uses competitive allele-specific PCR and a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS) that can unambiguously determine the presence or absence of the gene variant by displaying visible blue lines on the chromatographic printed-array strip. Notably, the results of our STH-PAS method were in 100% agreement with those obtained using standard Sanger sequencing and KASP assay genotyping methods for CYP4F2 gene deletion. Moreover, the results were obtained within 90 min, including the PCR amplification and signal detection processes. The sensitive and rapid nature of this novel method make it ideal for clinical genetic testing to predict drug efficacy and toxicity, and in doing so will aid in the development of individualized medicine and better patient care.

Improvement of malaria diagnostic system based on acridine orange staining.

BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent. METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya. RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard. CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs.

First detection and molecular identification of Entamoeba bovis from Japanese cattle.

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.

Anisakis haemoglobin is a main antigen inducing strong and prolonged immunoreactions in rats.

Anisakis simplex larvae are well known to cause gastrointestinal and allergic manifestations after ingestion of parasitized raw or undercooked seafood. The antibody recognition dynamics against the components of Anisakis larval antigen after primary and re-infection with Anisakis live larvae remain unclear. For this study, immunoblot analyses of serum IgG, IgE, and IgM against Anisakis larval somatic extract were performed in rats that had been orally inoculated with A. simplex live larvae. Multiple antigen fractions were recognized after primary infection. Their reaction was enhanced after re-infection. Antibody recognition was observed for 12 weeks after re-infection. The fraction of approximately 35 kDa contained a main antigen that induced strong and prolonged immunoreactions in IgG and IgE. The antibody reaction to this fraction appeared to be enhanced after inoculation of larval homogenates. This fraction was heat tolerant with boiling for 30 min. The fraction was spotted by immunoblotting after two-dimensional electrophoresis and was identified as Anisakis haemoglobin (Ani s 13) using mass spectrometry analysis. The amino acid sequences of haemoglobin mRNAs from two A. simplex sensu stricto and one Anisakis pegreffii were identified by RACE-PCR. They differed from those of two isolates of Pseudoterranova decipiens and A. pegreffii. Results of this study show that Anisakis haemoglobin, which is known to be a major allergen of A. simplex, induces strong and prolonged immunoreaction in rats. This report is the first to show the amino acid sequence variation of Anisakis haemoglobin mRNA between A. simplex sensu stricto and A. pegreffii.

High and Heterogeneous Prevalence of Asymptomatic and Sub-microscopic Malaria Infections on Islands in Lake Victoria, Kenya.

Kenya is intensifying its national efforts in malaria control to achieve malaria elimination. Detailed characterization of malaria infection among populations living in the areas where the disease is endemic in Kenya is a crucial priority, especially for planning and evaluating future malaria elimination strategy. This study aimed to investigate the distribution and extent of malaria infection on islands in Lake Victoria of Kenya to aid in designing new interventions for malaria elimination. Five cross-sectional surveys were conducted between January 2012 and August 2014 on four islands (Mfangano, Takawiri, Kibuogi and Ngodhe) in Lake Victoria and a coastal mainland (Ungoye). Malaria prevalence varied significantly among settings: highest in Ungoye, followed by the large island of Mfangano and lowest in the three remaining small islands. Of the 3867 malaria infections detected by PCR, 91.8% were asymptomatic, 50.3% were sub-microscopic, of which 94% were also asymptomatic. We observed geographical differences and age dependency in both proportion of sub-microscopic infections and asymptomatic parasite carriage. Our findings highlighted the local heterogeneity in malaria prevalence on islands and a coastal area in Lake Victoria, and provided support for the inclusion of mass drug administration as a component of the intervention package to eliminate malaria on islands.

Genetic analysis of Enterobius vermicularis isolated from a chimpanzee with lethal hemorrhagic colitis and pathology of the associated lesions.

Human pinworms, Enterobius vermicularis, are normally recognized as minor pathogens. However, a fatal case of human pinworm infection has been reported in a nonhuman primate, a zoo reared chimpanzee. Here, we histopathologically examined the lesions in tissues from the deceased chimpanzee and genetically characterized the isolated worms to investigate the pathogenicity and determine the phylogeny. We identified ulcers deep in the submucosa where many parasites were found to have invaded the lamina propria mucosa or submucous tissue. An inflammatory reaction consisting mainly of neutrophils and lymphocytes but not eosinophils was observed around the parasites, and intense hemorrhage in the lamina propria was confirmed. The parasites were morphologically similar to E. vermicularis based on the shape of the copulatory spicules. Mitochondrial cytochrome c oxidase subunit 1 gene products were amplified from worm DNA by PCR and were genetically identified as E. vermicularis based on >98.7% similarity of partial sequences. Phylogenetic analysis revealed that the sequences clustered together with other chimpanzee E. vermicularis isolates in a group which has been referred to as type C and which differs from human isolates (type A). The samples were negative for bacterial pathogens and Entamoeba histolytica indicating that E. vermicularis could be pathogenic in chimpanzees. Phylogenetic clustering of the isolates indicated that the parasite may be host specific.

Oral inoculation of live or dead third-stage larvae of Anisakis simplex in rats suggests that only live larvae induce production of antibody specific to A. simplex.

Live Anisakis simplex third-stage larvae (L3) penetrate gastrointestinal mucosa after they are ingested in raw or undercooked seafood, thereafter causing gastrointestinal manifestations and allergic manifestations such as urticaria and anaphylaxis. These allergic reactions are mediated by specific IgE to L3 allergens, especially excretory-secretory (ES) allergens. Recent evidences suggest that only live larvae can cause allergic reactions, although cases attributable to ingestion of cooked, frozen seafood have been reported. Therefore the risk of Anisakis-associated hypersensitivity by ingestion of properly cooked and frozen fish remains controversial. No prior report describes the kinetics of antibody production in experimental animals after oral inoculation with dead L3. This study used ELISA to assess antibody production in rats inoculated orally with dead L3. Positive absorbance value in IgG, IgM, and IgE specific to ES antigen from L3 were found in rats inoculated with live L3 but not with dead L3 (frozen, heated, cut, or homogenized). At one week post re-inoculation with live or frozen L3 to the initially sensitized rats, the absorbance value of the specific IgM and IgE to ES antigen elevated quickly and highly in rats that had been re-inoculated with live L3, but they decreased slightly or did not change in rats inoculated with frozen L3. These results suggest that only ingestion of live L3 can produce the specific antibody and induce initial and secondary sensitizations to L3.

Molecular evidence for person-to-person transmission of a novel subtype in Giardia duodenalis assemblage B at the rehabilitation institution for developmentally disabled people.

Giardia duodenalis was found in three patients and a health care worker at a rehabilitation institution for developmentally disabled people. The four isolates were genotyped and subtyped by multilocus homology searching and phylogenetic analyses of the following four loci: glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and ß-giardin (bg) as variable loci, and elongation factor 1 alpha (ef1α) as a conserved locus. The partial sequences, gdh (709 bp), tpi (526 bp), bg (724 bp), and ef1α (680 bp) of four isolates obtained were mutually identical, and the isolates were found to be a novel subtype in sub-assemblage BIV, strongly indicating that person-to-person transmission by a single subtype occurred at the institution.