Far-East Asian Toxoplasma isolates share ancestry with North and South/Central American recombinant lineages.

Toxoplasma gondii is a global protozoan pathogen. Clonal lineages predominate in Europe, North America, Africa, and China, whereas highly recombinant parasites are endemic in South/Central America. Far East Asian T. gondii isolates are not included in current global population genetic structure analyses at WGS resolution. Here we report a genome-wide population study that compared eight Japanese and two Chinese isolates against representative worldwide T. gondii genomes using POPSICLE, a novel population structure analyzing software. Also included were 7 genomes resurrected from non-viable isolates by target enrichment sequencing. Visualization of the genome structure by POPSICLE shows a mixture of Chinese haplogroup (HG) 13 haploblocks introgressed within the genomes of Japanese HG2 and North American HG12. Furthermore, two ancestral lineages were identified in the Japanese strains; one lineage shares a common ancestor with HG11 found in both Japanese strains and North American HG12. The other ancestral lineage, found in T. gondii isolates from a small island in Japan, is admixed with genetically diversified South/Central American strains. Taken together, this study suggests multiple ancestral links between Far East Asian and American T. gondii strains and provides insight into the transmission history of this cosmopolitan organism.

Acupuncture Treatment Improves Transport Stress in Microminipigs Through the Acupoint in Ears.

BACKGROUND/AIM: This study aimed to investigate the effects of acupuncture treatment through the ear acupoints on transport stress in experimental microminipigs. MATERIALS AND METHODS: Experiment 1: Six animals were equally divided into two groups (Control and Treatment). In the treatment group, before transportation (6 h; vehicle and plane), short, ultrathin circular transdermal needles were applied to locations corresponding to the acupoints on the apical area of both ears. Peripheral blood samples were collected from the cranial vena cava 2 days before and immediately after transportation. Blood stress markers, biochemistry indicators, and oxidative stress levels were examined. Experiment 2 (follow-up study: diarrhea incidence after transportation): Diarrhea incidence after transportation in the control and treatment groups was investigated. RESULTS: Experiment 1: Transport stress induced an increase in blood cortisol, serum amyloid A (SAA), glucose, non-esterified fatty acid, and derivatives of reactive oxygen metabolites (d-ROMs) and decreased the biological antioxidant potential (BAP)/d-ROMs ratio yet did not affect BAP. Acupuncture suppressed the increases in SAA and d-ROMs values and the decrease in BAP/d-ROMs ratio. Experiment 2: The total diarrhea incidence was 25% in the control group, whereas diarrhea was not observed in the treatment group. CONCLUSION: Acupuncture treatment suppresses hypothalamic-pituitary-adrenal function and, as a result, reduces transport stress without affecting the suppression of the central catecholaminergic system. Acupuncture treatment for transport stress can improve animal welfare.

In Vivo Metabolism of 1,5-Anhydro-d-fructose to 1,5-Anhydro-d-glucitol.

BACKGROUND/AIM: 1,5-Anhydro-d-fructose (1,5-AF, saccharide) and 1,5-anhydro-d-glucitol (1,5-AG) converted from 1,5-AF via the glycemic pathway have health benefits. However, this metabolism has not been sufficiently elucidated. To clarify the in vivo metabolism of 1,5-AF to 1,5-AG, porcine (blood kinetics) and human (urinary excretion) studies were conducted. MATERIALS AND METHODS: Microminipigs were administrated 1,5-AF orally or intravenously. Blood samples were obtained to analyse the kinetics of 1,5-AF and 1,5-AG. Urine samples were collected from human subjects who had orally ingested 1,5-AF, and the amounts of 1,5-AF and 1,5-AG excreted in the urine were analysed. RESULTS: In blood kinetics analysis, the time to the maximum concentration of 1,5-AF after intravenous administration was 0.5 h, whereas 1,5-AF was not observed after oral administration. The times to the maximum concentration of 1,5-AG after intravenous and oral administration were 1.5 h and 2 h, respectively. In urinary excretion, the concentration of 1,5-AG in urine rapidly increased after the administration of 1,5-AF, peaked at 2 h, whereas 1,5-AF was not detected. CONCLUSION: 1,5-AF was rapidly metabolized to 1.5-AG in vivo in swine and human.

In vitro antiviral activity of persimmon-derived tannin against avian influenza viruses.

Tannins derived from natural plant sources are known to provide many health benefits to humans and animals. Among the various tannins, those derived from persimmon (Diospyros kaki) have exhibited strong inactivating effects against pathogens that induce diseases in humans. However, few studies have focused on the antiviral effects of persimmon tannin against pathogen-induced diseases in animals. In this study, we investigated the antiviral effects of persimmon tannin against various avian influenza viruses revealing that tannin at a concentration of 1.0 mg ml-1 reduced viral infectivity in >6.0-log scale against all tested avian influenza viruses. In addition, this persimmon tannin concentration effectively inhibited the receptor binding and membrane fusion abilities of viral hemagglutinin (HA), which play important roles in avian influenza virus infection. These results suggest that persimmon tannin inactivates the HA of avian influenza viruses and reduces their infectivity. Persimmon tannin is a safer natural substance than the currently used chemical compound related to antiviral substance. When inactivation of the viruses which are present in environmental water such as roosting water of wild birds will be needed, persimmon tannin is expected to become an antiviral resource that may prevent the spread of several avian influenza virus subtypes.

Potential Biomarkers for Chronic Seasonal Heat Stress in Kagoshima Berkshire Pigs Reared in the Subtropical Region.

Introduction: Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons. Material and Methods: Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs' normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively. Results: The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts. Conclusion: The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

NR6A1 Allelic Frequencies as an Index for both Miniaturizing and Increasing Pig Body Size.

BACKGROUND/AIM: The number of vertebrae in swine varies from 19 to 23 and is associated with body size. Nuclear receptor subfamily 6 group A member 1 (NR6A1) is considered a strong candidate for affecting the number of vertebrae in swine. Wild boars, which uniformly have 19 vertebrae, have the wild type allele while multi-vertebrae European commercial pigs have the mutated allele. Our aim was to confirm the factor of the miniaturization. MATERIALS AND METHODS: We examined vertebrae number and NR6A1 polymorphism in the Microminipig and three domestic breeds that vary in body size. RESULTS: The Microminipig had 19 or less vertebrae and a wild type NR6A1 genotype. Three domestic breeds had more than 21 vertebrae while the largest vertebrae number was observed in multi-vertebrae-fixed Large White. Heterozygous genotypes were observed in the middle-sized indigenous pig while homozygous NR6A1 mutations were observed in European commercial breeds. CONCLUSION: NR6A1 could be a useful index for both miniaturizing and increasing pig body size.

Morphological and molecular identification of Eimeria spp. in breeding chicken farms of Japan.

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

Molecular and biological analysis revealed genetic diversity and high virulence strain of Toxoplasma gondii in Japan.

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol.

In vivo characterization of a Toxoplasma gondii strain TgCatJpTy1/k-3 isolated from a stray cat in Japan.

The Toxoplasma gondii strain TgCatJpTy1/k-3 (K-3), isolated from a stray cat in Tokyo, Japan, is categorized as a type II genotype. Since the K-3 strain is empirically known to form relatively larger cysts and exhibit weak pathogenesis in a mouse, it could serve as a useful model organism to study chronic T. gondii infection in the host. However, a detailed biological characterization of this strain had not been performed. In this study, we thoroughly assessed the K-3 strain in vivo using a mouse model. Tests indicated that pathogenicity of the K-3 strain was lower than that of the PLK strain, a clonal laboratory strain with a moderately pathogenic type II genotype. Further, cyst sizes of the K-3 strain were significantly larger than those of the PLK strain. Interestingly, K-3 cyst sizes in T. gondii-resistant ICR mice were larger than those in T. gondii-susceptible C57BL/6N mice. Our study suggests that the K-3 strain is suitable to study T. gondii cystogenesis and chronic infection, which are currently difficult to analyze using cell-adopted T. gondii strains.

Susceptibility to Various Coccidiostats in the Murine Coccidian Parasite Eimeria krijgsmanni.

INTRODUCTION: Murine Eimeria spp. have been used as effective experimental models of disease instead of large mammalian hosts such as cattle. We here examine drug susceptibility of the uncharacterized murine intestinal protozoan parasite, Eimeria krijgsmanni. MATERIALS AND METHODS: The effectiveness of different treatments against infection of E. krijgsmanni was examined for suppression of oocyst shedding: ST mixture ST mixture, pyrimethamine, Ektecin and toltrazuril. RESULTS: ST mixture and pyrimethamine did not suppress oocyst shedding effectively. Although therapeutic efficacy of Ektecin was demonstrated, the dose required was larger than that for cattle and chickens. Oocyst shedding was only completely suppressed completely by continuous administration of toltrazuril. Furthermore, it was confirmed through morphological examination that early developmental stage zoites appeared in host epithelial cells during and following treatment by toltrazuril, and toltrazuril could not eliminate residual zoites in epithelial cells. CONCLUSION: E. krijgsmanni may be relatively resistant to these anti-coccidian agents and might therefore have different characteristics that differ from other coccidia with regard to drug susceptibility.

Atypical virulence in a type III Toxoplasma gondii strain isolated in Japan.

The virulence of a type III Toxoplasma gondii strain isolated in Japan and designated here as TgCatJpGi1/TaJ was examined in mice and micro minipigs in this study. Despite its type III genotype, oral or intraperitoneal inoculation of cysts from it resulted in severe virulence in C57BL/6J and BALB/c mice. In contrast, mice inoculated with a high dose of TgCatJpGi1/TaJ tachyzoites showed no obvious clinical signs of infection, and all of them survived for >21 days post-inoculation. Furthermore, no clinical signs of infection were seen when micro minipigs were inoculated with 900 cysts. Interestingly, our allelic type screening of the virulence-related rop5, rop16, rop17, and rop18 genes, as based on restriction fragment length polymorphism analysis (RFLP), revealed that the RFLP patterns for TgCatJpGi1/TaJ were identical to those from nonvirulent type III parasites. These results suggest that TgCatJpGi1/TaJ possesses an unknown virulence factor or factors.

The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis.

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10 days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.

Characterization and expression analysis of a newly identified glutathione S-transferase of the hard tick Haemaphysalis longicornis during blood-feeding.

BACKGROUND: Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress. METHODS: Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT). RESULTS: We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed K m values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has K m values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5-8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands. CONCLUSIONS: In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress.

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.

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.

Detection and molecular characterization of Babesia, Theileria, and Hepatozoon species in hard ticks collected from Kagoshima, the southern region in Japan.

To reveal the distribution of tick-borne parasites, we established a novel nested polymerase chain reaction (PCR) system to detect the most common agents of tick-borne parasitic diseases, namely Babesia, Theileria, and Hepatozoon parasites. We collected host-seeking or animal-feeding ticks in Kagoshima Prefecture, the southernmost region of Kyusyu Island in southwestern Japan. Twenty of the total of 776 tick samples displayed a specific band of the appropriate size (approximately 1.4-1.6kbp) for the 18S rRNA genes in the novel nested PCR (20/776: 2.58%). These PCR products have individual sequences of Babesia spp. (from 8 ticks), Theileria spp. (from 9 ticks: one tick sample including at least two Theileria spp. sequences), and Hepatozoon spp. (from 3 ticks). Phylogenetic analyses revealed that these sequences were close to those of undescribed Babesia spp. detected in feral raccoons in Japan (5 sequences; 3 sequences being identical), Babesia gibsoni-like parasites detected in pigs in China (3 sequences; all sequences being identical), Theileria spp. detected in sika deer in Japan and China (10 sequences; 2 sequences being identical), Hepatozoon canis (one sequence), and Hepatozoon spp. detected in Japanese martens in Japan (two sequences). In summary, we showed that various tick-borne parasites exist in Kagoshima, the southern region in Japan by using the novel nested PCR system. These including undescribed species such as Babesia gibsoni-like parasites previously detected in pigs in China. Importantly, our results revealed new combinations of ticks and protozoan parasites in southern Japan. The results of this study will aid in the recognition of potential parasitic animal diseases caused by tick-borne parasites.

Report of fatal mixed infection with Cryptosporidium parvum and Giardia intestinalis in neonatal calves.

In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.

Dynamics of Theileria orientalis genotype population in cattle in a year-round grazing system.

Theirelia orientalis is a tick-borne haemoprotozoan parasite, and infection with this parasite is one of the most important diseases for grazing cattle. Co-infection of cattle with different genotypes of T. orientalis often occurs. In this study, we investigated the temporal dynamics of genotypes in cattle in a year-round grazing system in Japan. Genotype-specific PCR assays to determine major piroplasm surface protein (MPSP) genotypes (types 1 to 5) of T. orientalis were performed by using time-course blood samples collected from grazing cattle and ticks in a pasture. All 20 cattle investigated in this study were infected with T. orientalis. By using genotype-specific PCR, we detected the combination of genotypes of T. orientalis (types 1 to 5) from each cattle. These multiple genotypes of T. orientalis were also confirmed in ticks. Notably, each genotype of T. orientalis in cattle was temporally detected from cattle and more variable genotypes were found in summer. The observed temporal dynamics of the MPSP genotypes of T. orientalis in cattle could be explained by host immunity against the parasites or genetic recombination of parasite in ticks.

Role of the tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) from the hard tick Haemaphysalis longicornis in immunity against bacterial infection.

A tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) is a negative feedback regulator that controls excessive immune responses in vertebrates. The sequence of tick hemolymph TRAFD1 from the hard tick Haemaphysalis longicornis (HlTRAFD1) was analyzed after identification and cloning from the expressed sequence tag database. RT-PCR and Western blot analyses showed that HlTRAFD1 transcript and protein levels after blood feeding were present in all developmental stages, and the transcript level was consistently high in all organs examined from adult female ticks upon engorgement. Knockdown of HlTRAFD1 gene by RNA interference did not affect blood feeding or oviposition. However, HlTRAFD1 silencing affected the expression of the longicin gene, a defensin-like molecule, but not the lysozyme gene. Moreover, the survival rate of HlTRAFD1-silenced ticks was lower, and the number of E. coli was higher in the hemolymph and plasmatocytes after E. coli injection compared to the control group. These results suggested that HlTRAFD1 strongly affected both the humoral and cellular immunity of ticks.

Course of induced infection by Eimeria krijgsmannni in immunocompetent and immunodeficient mice.

Recently, we have demonstrated the utility of Eimeria krijgsmanni as a novel mouse eimerian parasite for elucidating the biological diversity. The parasite showed notable infectivity to mice with various levels of immune status and susceptibility to antimicrobial agents including coccidiostat. However, the detailed lifecycle of E. krijgsmanni had not yet been determined and this information was lacking in discussion of previous findings. In the present study, we clarified the morphological characteristics of E. krijgsmanni and its lifecycle in normal mice, and examined the effects in immunodeficient mice and lifecycle stage for challenge infections after the primary inoculation. In immunocompetent mice, the lifecycle consisted of four asexual stages and the sexual sages followed by formation of oocysts during the prepatent periods. Interestingly, the second-generation meronts were detected in all observation periods after the disappearance of the other stages. For the challenge infection of immunodeficient mice, all developmental stages except for the second generation meronts were temporarily vanished. This finding suggests a "rest" or marked delay in development and a "restart" of the promotion toward the next generations. The second generation meronts may play an important role in the lifecycle of E. krijgsmanni.