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Cryptosporidium is a genus of parasitic protozoa known to cause diarrheal disease that impacts both humans and animals through infection of various vertebrate species. Bats are recognized as reservoirs for zoonotic pathogens, including Cryptosporidium . The Philippines, renowned for its rich biodiversity, is home to diverse bat species, providing a unique ecological setting to investigate Cryptosporidium infection dynamics. Understanding the prevalence and genetic diversity of Cryptosporidium in Philippine bats is crucial for assessing their potential role in zoonotic disease transmission and associated public health risks. We investigated the prevalence and genotypic diversity of Cryptosporidium in bats in the Philippines. From January 2019 to March 2024, a total of 569 bats were captured and analyzed, with 14 of the bat samples testing positive for the 18 s rRNA gene of Cryptosporidium , yielding an overall infection rate of 2.46 %. One sample exhibited co-infection, with 18 s rRNA sequence analysis indicating mixed infection with a species closely related to Cryptosporidium parvum (intestinal Cryptosporidium ) and Cryptosporidium sp. (gastric Cryptosporidium ). Phylogenetic analysis of the 18S rRNA gene revealed that intestinal and gastric Cryptosporidium spp. form two distinct clades. Intestinal Cryptosporidium includes C. parvum , C. hominis , and most bat genotypes, while gastric Cryptosporidium , such as C. andersoni and C. serpentis , is typically found in reptiles and cattle. An unidentified Cryptosporidium species was also detected in one sample, whose sequence matched that of Cryptosporidium previously isolated from a human patient with diarrhea. Nine other samples exhibited genotypes related to C. parvum , indicating a potential for transmission to humans. The remaining three samples exhibited Cryptosporidium bat genotypes II and VI, which have previously been detected in Philippine bats. Our findings underscore the role of bats in the Philippines as potential reservoirs for Cryptosporidium and highlight the diversity of Cryptosporidium species in Philippine bats., Competing Interests: All authors declare no competing financial interests., (© 2024 The Author(s).)

We investigated intraerythrocytic Babesia parasites in 21 Japanese wild boars, Sus scrofa leucomystax , captured in Wakayama Prefecture on the mainland from 2008 to 2009 and in 31 Japanese wild boars from 2011 to 2013 in Kochi Prefecture on Shikoku Island, Japan. We detected small subunit ribosomal RNA (18S rRNA) gene (SSUrDNA) fragments of a Babesia species in 17 boars from Wakayama and 18 boars from Kochi. The nearly full SSUrDNA sequence (1669 bps) of this species was determined. A FASTA search revealed that the SSUrDNA sequence of the Babesia sp. in Japanese wild boars was the most homologous to those of several Babesia isolates reported as Babesia gibsoni . Phylogenetic analysis showed that the Babesia sp. found in Japanese wild boars was the closest relative to B. gibsoni but made a different clade from B. gibsoni . The Babesia sp. in Japanese wild boars was completely different from Babesia sp. Suis found in a European domestic pig, Sus scrofa domesticus . By microscopic examination, ring-shaped, oval and pear-shaped small sized intraerythrocytic parasites were observed on blood smears of 12 of 18 Japanese wild boars whose blood smears could be examined in Wakayama. We also detected SSUrDNA fragments of a Hepatozoon species in 6 of the 21 wild boars from Wakayama. The nearly full SSUrDNA sequence (1774 bps) of the Hepatozoon sp. was shown to be identical to that of Hepatozoon apri ., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Atsuko Saito-Ito reports financial support was provided by 10.13039/100007449Takeda Science Foundation, Japan. Atsuko Saito-Ito reports financial support was provided by Shinryokukai General incorporated Assocation, Japan. Motoko Nagano-Fujii reports financial support was provided by Hyogo University of Health Sciences Grant for Research Promotion, Japan. Motoko Nagano-Fujii reports financial support was provided by Joint research program of 10.13039/100019568Biosignal Research Center, Kobe University, Japan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector., (© 2024. The Author(s).)

Extra-gastrointestinal anisakidosis is rare. We herein report an Anisakis pegreffii infection in a patient with hepatic anisakidosis diagnosed based on its molecular identification. A 71-year-old male patient had a hepatic tumor presenting as a low-density area of 20 mm in diameter in segment 6 of the liver on abdominal ultrasonography, computed tomography, and magnetic resonance imaging. The surgically resected pathological specimen revealed a necrotizing eosinophilic granuloma containing nematode larvae, possibly an Anisakis larva. Molecular and phylogenetic analysis demonstrated Anisakis larvae belonging to A. pegreffii. The present results will help identify and characterize unknown Anisakis species in histological sections.

Cryptosporidium parvum is an apicomplexan parasite that causes severe zoonotic diarrhea in humans and calves. Since there are no effective treatments or vaccines for infants or immunocompromised patients, it is important to understand the molecular mechanisms of the parasite-host interaction for novel drug discovery. Mitogen-activated protein kinase (MAP kinase) is a key host factor in interactions between host and various pathogens, including parasites. Although the function of conventional MAP kinases against parasite infection has been investigated, that of atypical MAP kinases remains largely unknown. Therefore, we focused on one of the atypical MAP kinases, MAPK4, and its effect on C. parvum infection in human intestinal cells. Here, we report that MAPK4-deficient intestinal cells showed a significant reduction in C. parvum infection. We also show that host MAPK4 has a role in host cell survival from C. parvum infection. In addition, we show that C. parvum requires host MAPK4 for its successful invasion and asexual reproduction. Taken together, our data suggest that MAPK4 is an important host factor contributing to C. parvum infection in human intestinal cells., (© 2023. The Author(s).)

Purpose: This study compared concentric-eccentric coupled (CON-ECC), concentric-only (CON), and eccentric-only (ECC) resistance training of the elbow flexors for their effects on muscle strength and hypertrophy., Methods: Non-resistance-trained young adults were assigned to one of the four groups: CON-ECC (n = 14), CON (n = 14) and ECC (n = 14) training groups, and a control group (n = 11) that had measurements only. The training group participants performed dominant arm elbow flexor resistance training in extended elbow joint angles (0°-50°) twice a week for 5 weeks. The total training volume (dumbbell weight × number of contractions) in CON-ECC (5745 ± 1020 kg) was double of that in CON (2930 ± 859 kg) and ECC (3035 ± 844 kg), because 3 sets of 10 contractions were performed for both directions in CON-ECC. Maximum voluntary isometric (MVC-ISO), concentric (MVC-CON), and eccentric contraction (MVC-ECC) torque of the elbow flexors and biceps brachii and brachialis muscle thickness (MT) were measured at baseline, and 3-9 days post-last training session., Results: No significant changes in any measures were evident for the control group. The CON-ECC and ECC groups showed increases (P < 0.05) in MVC-ISO (12.0 ± 15.7% and 11.3 ± 10.8%, respectively) and MVC-ECC torque (12.5 ± 18.3%, 16.2 ± 11.0%) similarly. Increases in MVC-CON torque (P < 0.05) were evident for the CON-ECC (17.5 ± 13.5%), CON (10.5 ± 12.8%), and ECC (14.2 ± 10.4%) groups without a significant difference among groups. MT increased (P < 0.01) after CON-ECC (10.6 ± 5.4%) and ECC (9.7 ± 7.2%) similarly, but not significantly after CON (2.5 ± 4.8%)., Conclusions: ECC training increased muscle strength and thickness similarly to CON-ECC training, despite the half training volume, suggesting that concentric contractions contributed little to the training effects., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Our previous study found that one maximal voluntary eccentric contraction (MVC-ECC) performed daily for 5 days a week for 4 weeks increased MVC-ECC, isometric (MVC-ISO), and concentric contraction (MVC-CON) torque of the elbow flexors more than 10%. The present study investigated the effects of six maximal voluntary eccentric contractions on the MVC torques and biceps brachii and brachialis muscle thickness (MT). Thirty-six healthy young adults were placed to one of the three groups (N = 12 per group); the 6 × 1 group that performed one set of six contractions once a week, the 6 × 5 group that performed one set of six contractions a day for 5 days a week, and the 30 × 1 group that performed five sets of six contractions a day in a week. The training duration was 4 weeks for all groups, and changes in MVC-ECC, MVC-CON and MVC-ISO torque, and MT before and after the 4-week training were compared among the groups. The 6 × 1 group did not show significant changes in muscle strength and MT. Significant (p < 0.05) increases in MVC-ECC (13.5 ± 11.5%), MVC-ISO (9.3 ± 5.5%), MVC-CON torque (11.1 ± 7.4%) were evident for the 6 × 5 group only, and increases in MT were found for the 6 × 5 (10.4 ± 4.4%) and 30 × 1 (8.0 ± 5.8%) groups without a significant difference. These results suggest that performing a small number of eccentric contractions 5 days a week is more effective for increasing muscle strength than performing a larger volume of eccentric contractions once a week. However, it appears that training volume is a factor for muscle hypertrophy in a short-term training., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The present study compared a 3-s isometric maximal voluntary contraction (MVC), concentric MVC and eccentric MVC of the elbow flexors performed daily for 5 days a week for 4 weeks for changes in muscle strength and thickness. Young sedentary individuals were assigned to one of three training groups (n = 13 per group) that performed either 3-s isometric, concentric, or eccentric MVC once a day for 20 days, or to a control group (n = 10) that had measurements without training. The participants in the isometric group performed isometric MVC at 55° (0.96 rad) elbow flexion, and those in the concentric or eccentric group performed concentric MVC or eccentric MVC between 10° (0.17 rad) and 100° (1.75 rad) elbow flexion at 30°/s (0.52 rad/s) on an isokinetic dynamometer. MVC isometric torque at 20° (0.35 rad), 55° (0.96 rad), and 90° (1.57 rad) elbow flexion, MVC concentric and eccentric torque at 30°/s (0.52 rad/s) and 180°/s (3.14 rad/s), and muscle thickness (MT) of biceps brachii and brachialis were measured before and several days after the 20th exercise session. The control group did not show any changes. The eccentric group showed increases (p < 0.01) in isometric (three angle average: 10.2 ± 6.4%), concentric (two velocity average: 12.8 ± 9.6%), and eccentric MVC torque (12.2 ± 7.8%). An increase (p < 0.05) was limited for isometric MVC torque (6.3 ± 6.0%) in the concentric group, and for eccentric MVC torque (7.2 ± 4.4%) in the isometric group. No significant changes in MT were evident for all groups. Performing one 3-s MVC a day increased muscle strength, but eccentric MVC produced more potent effects than isometric or concentric MVC., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods., Methods: We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates., Results: The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates., Conclusion: This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology., (© 2021. The Author(s).)

Cryptosporidium and Toxoplasma are parasites that have caused problems worldwide. Cryptosporidium causes severe watery diarrhoea and may be fatal in immunocompromised patients and in infants. Nitazoxanide is the only agent currently approved by the FDA, but its efficacy is limited. Toxoplasmosis is also a problem in the immunocompromised, as currently available treatment options have limited efficacy and patient tolerance can be poor. In the present investigation, we screened libraries of epigenetic compounds to identify those that inhibited C. parvum growth. Nullscript was identified as a compound with an inhibitory effect on C. parvum and T. gondii growth, and was less toxic to host cells. Nullscript was also able to significantly decrease oocyst excretion in C. parvum-infected SCID mice., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cryptosporidium spp. are enteric protozoan parasites that infect a wide range of hosts including humans, and domestic and wild animals. The aim of this study was to molecularly characterize the Cryptosporidium spp. found in calf faeces in Japan. A total of 80 pre-weaned beef and dairy calves' diarrhoeic faecal specimens were collected from nine different prefectures in Japan. A nested polymerase chain reaction targeting the small subunit 18S rRNA and GP60 genes were used to detect the Cryptosporidium genotypes and subtypes. 83.8% (67 out of 80) of the specimens were positive for Cryptosporidium spp.; Cryptosporidium was found in both beef and dairy calves. Cryptosporidium parvum was the predominant species, detected in 77.5% (31/40) of beef calves and 80% (32/40) of dairy calves. Cryptosporidium bovis was also detected, 5.0% (2/40) of dairy calves, and C. ryanae was also found 2.5% (1/40) of dairy calves. One mixed-species infection, 2.5% (1/40) was detected in a beef calf having C. parvum, and C. ryanae. We detected the most common subtype of C. parvum (i.e., IIaA15G2R1), as well as other subtypes (i.e., IIaA14G3R1, IIaA14G2R1, and IIaA13G1R1) that have not previously been detected in calves in Japan. Our results demonstrate the widespread diversity of Cryptosporidium infection in calves in Japan., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cryptosporidium is an obligate intracellular parasite which can cause fatal diarrheal disease in exotic animals. Sugar gliders (Petaurus breviceps), hedgehogs (Atelerix albiventris), chinchillas (Chinchilla lanigera), and common leopard geckos (Eublepharis macularius) are popular exotic animals commonly sold in pet shops in Japan. We herein investigated the species and subtypes of Cryptosporidium in these animals. Cryptosporidium fayeri was detected in a sugar glider in a Japanese animal hospital. Sequence analyses of the 60-kDa glycoprotein (gp60) gene revealed that C. fayeri belonged to subtype family IVh (IVhA13G2T1), which was proposed to be a new subtype. This is the first study to report C. fayeri infection in a sugar glider. In other animals, the Cryptosporidium horse genotype, C. ubiquitum, and C. varanii were detected in two four-toed hedgehogs (A. albiventris), a chinchilla (C. lanigera), and common leopard gecko (E. macularius), respectively. The gp60 subtypes identified were VIbA13 of the horse genotype and XIId of C. ubiquitum. The present results revealed that potentially zoonotic Cryptosporidium is widespread in exotic animals in Japan., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cryptosporidium is an opportunistic parasite that has been reported in >30 avian hosts worldwide, however, there is no information regarding Cryptosporidium spp. in poultry in Bangladesh. Accordingly, we investigated the prevalence of Cryptosporidium spp. in poultry at open live bird markets in Bangladesh. A total of 197 samples were randomly collected from poultry at open live bird markets in Bangladesh and screened for the detection of Cryptosporidium. Initial microscopic examination revealed Cryptosporidium spp. was observed in 19.8% (39/197) of the poultry specimens. Subsequent nested PCR targeting the 18S rRNA gene revealed that 15.7% (31/197) of the samples were Cryptosporidium positive. Of these 31 samples, 17 were Cryptosporidium baileyi (8.7%), 12 were Cryptosporidium meleagridis (6.0%), and 2 were Cryptosporidium parvum (1.0%). Nucleotide sequence analysis of the GP60 gene of the C. meleagridis revealed that two subtypes (IIIbA21G1R1 and IIIbA23G1R1), which were found in broiler, native and sonali chickens and a pigeon, matched those previously reported in humans and poultry. We identified two novel subtypes (IIIbA21G2R1 and IIIbA20G2R1) in sonali chickens, a broiler chicken and a layer chicken. We also amplified the GP60 gene of C. parvum and found two subtypes (IIaA11G2R1 and IIaA13G2R1) in a sonali and a broiler chicken that were previously reported in calf. These findings suggest that poultry can be a source of cryptosporidial infections for humans and animals in Bangladesh. This is the first molecular investigation of Cryptosporidium genotypes and subtypes in poultry at open live bird markets in Bangladesh., Competing Interests: The authors have no conflicts of interest to declare., (© 2020 The Authors.)

The faecal microbiota plays a critical role in host health, with alterations in the human faecal microbial composition associated with various conditions, particularly diarrhoeal diseases. However, little is known about microbial changes during cryptosporidiosis, one of the most important diarrhoeal diseases caused by protozoa in cattle. In this study, alterations in the faecal microbiota of neonatal calves as a result of Cryptosporidium parvum infection were investigated on a C. parvum-positive farm. Comparisons were made among groups of C. parvum-infected, rotavirus-infected, and the pathogen-negative calves. A specific increase in the abundance of Fusobacterium was observed in the faecal microbiota of C. parvum-infected animals. Diarrhoea severity increased in accordance with the abundance of C. parvum and Fusobacterium. Moreover, the specific increase of Fusobacterium appeared to be a universal feature of C. parvum infection, since neonatal calves from geographically separated areas showed the same result. These observations indicated that the growth of Fusobacterium may be an important aggravating factor of cryptosporidiosis.

Bats are potential reservoirs of Cryptosporidium and Eimeria. The genus Cryptosporidium infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Many epidemiological studies in wild animals have been performed; however, most of them relied on only PCR-based detection because of the difficulty of performing pathological analyses. Accordingly, the natural host and pathogenicity of Cryptosporidium bat genotypes remain unclear. In this study, we captured Eptesicus nilssonii (Northern bats) in Hokkaido, Japan. Of the three intestinal samples obtained, two were positive for Cryptosporidium spp. and one was positive for Eimeria spp. The corresponding microorganisms were also confirmed histopathologically. We detected the novel Cryptosporidium bat genotype XII and Eimeria rioarribaensis in bat intestine.

Toxoplasma gondii rhoptry neck protein 4 (TgRON4) is a component of the moving junction, a key structure for host cell invasion. We previously showed that host cellular β-tubulin is a binding partner of TgRON4 in the invasion process. Here, to identify other binding partners of TgRON4 in the host cell, we examined the binding of TgRON4 to components of the host cell surface. TgRON4 binds to various mammalian cells, but this binding disappeared in glycosaminoglycan- and heparan sulfate-deficient CHO cells and after heparitinase treatment of mammalian cells. The C-terminal half of TgRON4 showed relatively strong binding to cells and heparin agarose. A glycoarray assay indicated that TgRON4 binds to heparin and modified heparin derivatives. Immunoprecipitation of T. gondii-infected CHO cell lysates showed that TgRON4 interacts with glypican 1 during Toxoplasma invasion. This interaction suggests a role for heparan sulfate in parasite invasion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0-27.2)×24.7 (23.4-26.0) μm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5-20.5) and 11.2 (10.2-12.1) μm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Understanding the molecular defense mechanism of macrophages and identifying their effector molecules against malarial parasites may provide important clues for the discovery of new therapies. To analyze the immunological responses of malarial parasite-induced macrophages, we used DNA microarray technology to examine the gene profile of differentiated macrophages phagocytizing Plasmodium falciparum-parasitized erythrocytes (iRBC). The transcriptional gene profile of macrophages in response to iRBCs represented 168 down-regulated genes, which were mainly involved in the cellular immune response, and 216 upregulated genes, which were involved in cellular proteolysis, growth, and adhesion. Importantly, the specific upregulation of β-defensin 130 (DEFB130) in these macrophages suggested a possible role for DEFB130 in malarial parasite elimination. Differentiated macrophages phagocytizing iRBCs exhibited an increase in intracellular DEFB130 levels and DEFB130 appeared to accumulate at the site of iRBC engulfment. Transfection of esiRNA-mediated knockdown of DEFB130 into macrophages resulted in a remarkable reduction in their antiplasmodial activity in vitro. Furthermore, DEFB130 synthetic peptide exhibited a modest toxic effect on P. falciparum in vitro and P. yoelii in vivo, unlike scrambled DEFB130 peptide, which showed no antiplasmodial activity. Together, these results suggest that DEFB130 might be one of the macrophage effector molecules for eliminating malarial parasites. Our data broaden our knowledge of the immunological response of macrophages to iRBCs and shed light on a new target for therapeutic intervention., Competing Interests: The authors declare no competing financial interests.

Unlabelled: Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals., Importance: Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that TgPKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation., (Copyright © 2016 Sugi et al.)

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Bats are naturally infected with zoonotic pathogens; thus, they are potential reservoirs of parasites. We investigated the species and genotype distribution as well as prevalence of Cryptosporidium and Eimeria in Philippine bats. We captured and examined 45 bats; four were positive for Cryptosporidium spp. and seven were positive for Eimeria spp. We detected Cryptosporidium bat genotype II from Ptenochirus jagori. Three other Cryptosporidium sequences, detected from Rhinolophus inops, Cynopterus brachyotis, and Eonycteris spelaea, could not be classified as any known species or genotype; we therefore propose the novel genotype Cryptosporidium bat genotypes V, VI, and VII. Bat genotype V is associated with human cryptosporidiosis clade, and therefore, this genotype may be transmissible to humans. Among the Eimeria sequences, BE3 detected from Scotophilus kuhlii was classified with known bat and rodent clades; however, other sequences detected from C. brachyotis, E. spelaea, Rousettus amplexicaudatus, and R. inops could not be classified with known Eimeria species. These isolates might represent a new genotype. Our findings demonstrate that the bats of the Philippines represent a reservoir of multiple Cryptosporidium and Eimeria spp.

We show that Cryptosporidium parvum virus 1 (CSpV1), a member of the family Partitiviridae, genus Cryspovirus that can infect Cryptosporidium parvum, is a new candidate for high-resolution tool for tracing C. parvum. CSpV1 was detected in all C. parvum-positive samples tested. Phylogenetic analysis of dsRNA1 sequence from CSpV1 can distinguish infected areas of C. parvum on the national level. Sequences detected in samples from Iwate prefecture and other islands (Tanegashima, and Okinawa) belonged to a single clade. This system can differentiate the samples from Hokkaido and south part of Japan as well as from other countries. Samples from Iwate, Tanegashima, and Okinawa belonged to a single subclade, respectively. Therefore, the CSpV1 dsRNA sequences reflect the regional distribution of their host and have potential as a high-resolution tool to trace C. parvum IIaA15G2R1 subtype., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cryptosporidium parvum is an apicomplexan parasite that can cause serious watery diarrhea, cryptosporidiosis, in human and other mammals. C. parvum invades gastrointestinal epithelial cells, which have abundant glycosaminoglycans on their cell surface. However, little is known about the interaction between C. parvum and glycosaminoglycans. In this study, we assessed the inhibitory effect of sulfated polysaccharides on C. parvum invasion of host cells and identified the parasite ligands that interact with sulfated polysaccharides. Among five sulfated polysaccharides tested, heparin had the highest, dose-dependent inhibitory effect on parasite invasion. Heparan sulfate-deficient cells were less susceptible to C. parvum infection. We further identified 31 parasite proteins that potentially interact with heparin. Of these, we confirmed that C. parvum elongation factor 1α (CpEF1α), which plays a role in C. parvum invasion, binds to heparin and to the surface of HCT-8 cells. Our results further our understanding of the molecular basis of C. parvum infection and will facilitate the development of anti-cryptosporidial agents.

Background: There is an urgent need to develop and test novel compounds against malaria infection. Carrageenans, sulphated polysaccharides derived from seaweeds, have been previously shown to inhibit Plasmodium falciparum in vitro. However, they are inflammatory and alter the permeability of the blood-brain barrier, raising concerns that their use as a treatment for malaria could lead to cerebral malaria (CM), a severe complication of the disease. In this work, the authors look into the effects of the administration of λ-carrageenan to the development and severity of CM in BALB/c mice, a relatively non-susceptible model, during infection with the ANKA strain of Plasmodium berghei., Methods: Five-week-old female BALB/c mice were infected with P. berghei intraperitoneally. One group was treated with λ-carrageenan (PbCGN) following the 4-day suppressive test protocol, whereas the other group was not treated (PbN). Another group of healthy BALB/c mice was similarly given λ-carrageenan (CGN) for comparison. The following parameters were assessed: parasitaemia, clinical signs of CM, and mortality. Brain and other vital organs were collected and examined for gross and histopathological lesions. Evans blue dye assays were employed to assess blood-brain barrier integrity., Results: Plasmodium berghei ANKA-infected BALB/c mice treated with λ-carrageenan died earlier than those that received no treatment. Histopathological examination revealed that intracerebral haemorrhages related to CM were present in both groups of infected BALB/c mice, but were more numerous in those treated with λ-carrageenan than in mock-treated animals. Inflammatory lesions were also observed only in the λ-carrageenan-treated mice. These observations are consistent with the clinical signs associated with CM, such as head tilt, convulsions, and coma, which were observed only in this group, and may account for the earlier death of the mice., Conclusion: The results of this study indicate that the administration of λ-carrageenan exacerbates the severe brain lesions and clinical signs associated with CM in BALB/c mice infected with P. berghei ANKA.

Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC&#95;1C9, for a high-throughput assay. PLK/DLUC&#95;1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10(2) parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC&#95;1C9 expressed Renilla luciferase under the parasite's α-tubulin promoter. Renilla luciferase activity was detected with at least 10(2) parasites. By using PLK/DLUC&#95;1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The 23-kDa piroplasm membrane protein of Theileria orientalis (p23) is an immunogenic protein expressed during the intraerythrocytic stage of the parasite; its function, however, remains unclear. To evaluate the host factor or factors that interact with p23, we examined the binding of p23 to components of the host cell surface. Recombinant p23 protein of the Ikeda genotype failed to bind to bovine red blood cells or to peripheral blood mononuclear cells, but did bind to Madin-Darby Bovine Kidney (MDBK) cells. A glycoarray assay showed that recombinant p23 proteins from the three genotypes bound to heparin, indicating that p23 is a heparin-binding Theileria surface molecule. Further analysis of heparin-binding molecules is useful for understanding attachment and invasion of T. orientalis merozoites.

Here, we assessed the sulfated derivative of the microbial polysaccharide gellan gum and derivatives of λ and κ-carrageenans for their ability to inhibit Plasmodium falciparum 3D7 and Dd2 growth and invasion of red blood cells in vitro. Growth inhibition was assessed by means of flow cytometry after a 96-h exposure to the inhibitors and invasion inhibition was assessed by counting ring parasites after a 20-h exposure to them. Gellan sulfate strongly inhibited invasion and modestly inhibited growth for both P. falciparum 3D7 and Dd2; both inhibitory effects exceeded those achieved with native gellan gum. The hydrolyzed λ-carrageenan and oversulfated κ-carrageenan were less inhibitory than their native forms. In vitro cytotoxicity and anticoagulation assays performed to determine the suitability of the modified polysaccharides for in vivo studies showed that our synthesized gellan sulfate had low cytotoxicity and anticoagulant activity.

The intracellular parasite Toxoplasma gondii invades almost all nucleated cells, and has infected approximately 34% of the world's population to date. In order to develop effective vaccines against T. gondii infection, understanding of the role of the molecules that are involved in the invasion process is important. For this purpose, we characterized T. gondii proteins that contain microneme adhesive repeats (MARs), which are common in moving junction proteins. T. gondii MAR domain-containing protein 4a (TgMCP4a), which contains repeats of 17-22 amino acid segments at the N-terminus and three putative MAR domains at the C-terminus, is localized near the rhoptry of extracellular parasites. Following infection, TgMCP4a was detected in the parasitophorous vacuole. The recombinant Fc-TgMCP4a N-terminus protein (rTgMCP4a-1/Fc) showed binding activity to the surface proteins of Vero, 293T, and CHO cells. The recombinant GST-TgMCP4a N-terminus protein (rTgMCP4a-1/GST), which exhibited binding activity, was used to pull down the interacting factors from 293T cell lysate, and subsequent mass spectrometry analysis revealed that three types of heat shock proteins (HSPs) interacted with TgMCP4a. Transfection of a FLAG fusion protein of TgMCP4a-1 (rTgMCP4a-1/FLAG) into 293T cell and the following immunoprecipitation with anti-FLAG antibody confirmed the interactions of HSC70 with TgMCP4a. The addition of rTgMCP4a-1/GST into the culture medium significantly affected the growth of the parasite. This study hints that T. gondii may employ HSP proteins of host cell to facilitate their growth., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Toxoplasma gondii is one of the most prevalent parasites, causing toxoplasmosis in various warm-blooded animals, including humans. Because of the broad range of hosts susceptible to T. gondii, it had been postulated that a universal component of the host cell surface, such as glycosaminoglycans (GAGs), may act as a receptor for T. gondii infection. Carruthers et al. (Infect Immun 68:4005-4011, 2000) showed that soluble GAGs have also been shown to disrupt parasite binding to human fibroblasts. Therefore, we investigated the inhibitory effect of GAGs and their analogue dextran sulfate (DS) on T. gondii infection. For up to 24 h of incubation after inoculation of T. gondii, the inhibitory effect of GAGs on T. gondii infection and growth inside the host cell was weak. In contrast, DS markedly inhibited T. gondii infection. Moreover, low molecular weight DS particularly slowed the growth of T. gondii inside host cells. DS10 (dextran sulfate MW 10 kDa) was the most effective agent in these in vitro experiments and was therefore tested for its inhibitory effects in animal experiments; infection inhibition by DS10 was confirmed under these in vivo conditions. In this report, we showed that DSs, especially DS10, have the potential of a new type of drug for toxoplasmosis.

Toxoplasma rhoptry neck protein 4 (TgRON4) is a component of the moving junction macromolecular complex that plays a central role during invasion. TgRON4 is exposed on the cytosolic side of the host cell during invasion, but its molecular interactions remain unclear. Here, we identified host cellular β-tubulin as a binding partner of TgRON4, but not Plasmodium RON4. Coimmunoprecipitation studies in mammalian cells demonstrated that the C-terminal 15-kDa region of β-tubulin was sufficient for binding to TgRON4, and that a 17-kDa region in the proximal C-terminus of TgRON4 was required for binding to the C-terminal region of β-tubulin. Analysis of T. gondii-infected lysates from CHO cells expressing the TgRON4-binding region showed that the C-terminal region of β-tubulin interacted with TgRON4 at early invasion step. Our results provide evidence for a parasite-specific interaction between TgRON4 and the host cell cytoskeleton in parasite-infected cells.

The cell cycle of Plasmodium is unique among major eukaryotic cell cycle models. Cyclin-dependent kinases (CDKs) are thought to be the key molecular switches that regulate cell cycle progression in the parasite. However, little information is available about Plasmodium CDKs. The present study was performed to investigate the effects of a CDK inhibitor, olomoucine, on the erythrocytic growth of Plasmodium falciparum. This agent inhibited the growth of the parasite at the trophozoite/schizont stage. Furthermore, we characterized the Plasmodium CDK homolog, P. falciparum cdc2-related kinase-1 (Pfcrk-1), which is a potential target of olomoucine. We synthesized a functional kinase domain of Pfcrk-1 as a GST fusion protein using a wheat germ protein expression system, and examined its phosphorylation activity. The activity of this catalytic domain was higher than that of GST-GFP control, but the same as that of a kinase-negative mutant of Pfcrk-1. After the phosphatase treatment, the labeling of [γ-(32)P]ATP was abolished. Recombinant human cyclin proteins were added to these kinase reactions, but there were no differences in activity. This report provides important information for the future investigation of Plasmodium CDKs., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Wild rodents are naturally infected with zoonotic Cryptosporidium; thus, they are potential reservoirs of the parasites. Mice are common rodents frequently found in agricultural areas and have many opportunities to contact other wild animals, livestock, and humans. Irrespective of the potential risk, there are few epidemiologic studies of Cryptosporidium in wild mice because of their low economic importance and the difficulty in conducting surveys. Hence, the species and genotypes of Cryptosporidium in wild mice living around various areas remain unclear. We investigated the species and genotype distribution and prevalence of Cryptosporidium in the large Japanese field mouse (Apodemus speciosus) in an agricultural site in Osaki, Miyagi Prefecture, Japan. In total, 15 mice were captured and examined in this study. By microscopic analysis, only one mouse (JFM 3) was determined to be Cryptosporidium-positive, while the parasite were detected in four mice (JFM 3, 6, 10, and 15) by a molecular approach using partial SSU rRNA gene sequences. Based on nucleotide sequence and phylogenetic analysis, the Cryptosporidium isolates were identified as C. ubiquitum (from JFM 10) and C. muris (from JFM 3 and 6). In contrast, the Cryptosporidium in JFM 15 was not identified as a known species or genotype and is therefore proposed as a novel genotype; the Naruko genotype. More molecular data are necessary to elucidate the taxonomic identity of this novel Cryptosporidium genotype. The C. muris Japanese field mouse genotypes showed marked divergence compared to that in a previous report. The large Japanese field mouse might thus represent a reservoir of multiple Cryptosporidium spp., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cattle are major hosts of Cryptosporidium. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. Fecal samples were collected from calves in Ishikari District, Hokkaido, Japan and examined by PCR and sequence analyses. Among the 107 fecal samples collected in May and June 2012, 25 (23%) were positive for Cryptosporidium, including 8 samples (7%) having C. parvum, 10 (9%) having C. bovis and 7 (7%) having C. ryanae. This is first time C. ryanae has been detected in Hokkaido. Furthermore, it is the first detection of C. ryanae from pre-weaned calves in Japan. Microscopic observation with the flotation method is powerful and traditional tool for screening for Cryptosporidium species, but it sometimes leads to low detection of Cryptosporidium with low oocyst shedding intensity. If calves with or without diarrhea are examined using the molecular diagnostic tools, C. bovis and C. ryanae might be detected in other areas of Japan including Hokkaido. Here, the zoonotic species, C. parvum, was also observed. Therefore, calves can be potential sources of cryptosporidial infections for humans and other animals. The detection of C. parvum was statistically correlated with diarrhea in calves.

Toxoplasma gondii is an important food and waterborne pathogen that causes severe disease in immunocompromised patients. Bumped kinase inhibitors (BKIs) have an antiparasitic effect on T. gondii tachyzoite growth by targeting T. gondii calmodulin-domain protein kinase 1 (TgCDPK1). To identify mutations that confer resistance to BKIs, chemical mutagenesis was performed, followed by selection in media containing either 250 or 1000 nM 1NM-PP1. Whole-genome sequence analysis of resistant clones revealed single nucleotide mutations in T. gondii mitogen-activated protein kinase 1 (TgMAPK1) at amino acids 162 (L162Q) and 171 (I171N). Plasmid constructs having the TgMAPK1 L162Q mutant sequence successfully replaced native TgMAPK1 genome locus in the presence of 1000 nM 1NM-PP1. The inhibitory effect of 1NM-PP1 on cell division observed in the parent clone was decreased in 1NM-PP1-resistant clones; however, effects on parasite invasion and calcium-induced egress were similar in both parent and resistant clones. A plasmid construct expressing the full length TgMAPK1 splicing isoform with L162Q mutation successfully complemented TgMAPK1 function in the pressure of 250 nM 1NM-PP1 in plaque assay. 1NM-PP1-resistant clones showed resistance to other BKIs (3MB-PP1 and 3BrB-PP1) with different levels. Here we identify TgMAPK1 as a novel target for 1NM-PP1 activity. This inhibitory effect is mediated through inhibition of tachyzoite cell division, and can be overcome through mutations at multiple residues in TgMAPK1.

Cattle are major hosts of Cryptosporidium spp. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. In many areas, cow-calf glazing system is an important beef cattle rearing method with distinct advantages in terms of cost and the labor required. However, few epidemiologic studies of Cryptosporidium spp. have been conducted in this system, especially using molecular diagnostic tools. To understand the transmission of Cryptosporidium spp. in a grazing system, we followed cryptosporidiosis on a grazing farm in Osaki City, Miyagi Prefecture, in northwest Japan for one year. Fecal samples were collected from Japanese Black and Japanese Shorthorn cattle and examined by PCR-RFLP and sequence analyses. Of 113 fecal samples collected in October 2010, 23 (20%) were positive for Cryptosporidium, including 15 samples (13%) having C. bovis, 6 (5%) having C. ryanae, and 2 (2%) having mixed infections of both species. Additionally, C. bovis or C. ryanae was detected on all other sampling dates involving smaller numbers of animals. The infection rate of C. bovis was significantly different among age groups, and calve-to-calve infection might be the major route of cryptosporidiosis transmission in beef cattle. Interestingly, one animal had C. bovis infection or re-infection for one year. Our results suggest that C. bovis and C. ryanae are distributed in Japan, but might have low level of detection in grazing beef cattle., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cryptosporidium parvum HNJ-1 is widely used as a reference strain in Japan. In the present study, the parasite was subjected for further molecular analysis including transcribed ribosomal region (ITS rRNA), dihydrofolate reductase (DHFR) and surface glycoprotein (GP60) genes. Partial sequence analysis of these genes indicated extensive polymorphism in ITS region compared with relevant sequences of other Cryptosporidium parvum isolates. In addition, this strain was identified as C. parvum IIaA15G2R1 subtype, based on the sequence results of GP60 gene locus.