Zoonotic human liver flukes, a type 1 biocarcinogen, in freshwater fishes: genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh.

BACKGROUND: Opisthorchiid flukes, particularly Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, and Metorchis spp. are the most common fish-borne zoonotic human liver flukes (hLFs). Liver fluke infections are more prevalent in resource-deprived and underprivileged areas. We herein estimated the prevalence of the metacercariae (MC) of major hLFs in common large freshwater fishes (lFWF) marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs. METHODS: The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (RFLP), sequencing, and bioinformatic analyses. The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared (χ2) test. RESULTS: The MC of C. sinensis, Opisthorchis spp., and Metorchis spp. were detected in 11 species of common and popular lFWF. In lFWF, the estimated prevalence was 18.7% and the mean load was 137.4 ± 149.8 MC per 100 g of fish. The prevalence was the highest (P < 0.05) in spotted snakehead fishes (Channa punctata, 63.6%). The highest rate of infection (P < 0.05) was observed with the MC of C. sinensis (11.8%). Metacercariae were almost equally (P > 0.05) distributed between the head and body of fishes. The infection rate was slightly higher in cultured (19.6%) fishes. The MC of C. sinensis, O. felineus, O. viverrini, and Metorchis orientalis in fishes were confirmed using PCR, PCR-RFLP and bioinformatics. The cercariae of opisthorchiid (Pleurolophocercus cercariae) flukes were only recovered from Bithynia spp. (3.9%, 42 out of 1089). The ova of hLFs from dogs (4.3%, 5 out of 116) and cats (6.0%, 6 out of 100), and adult flukes (M. orientalis) from ducks (41.1% 113 out of 275) were detected. CONCLUSIONS: The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh. Reservoir hosts, such as street dogs, cats, and ducks carried the patent infection, and residents of Bangladesh are at risk.

Receptor for Advanced Glycation End Product (RAGE) Modulates Inflammation During Feeding of the Hard Tick, Haemaphysalis longicornis in Mice.

Ticks are notorious blood-sucking ectoparasites that affect both humans and animals. They serve as a unique vector of various deadly diseases. Here, we have shown the roles of the receptor for advanced glycation end products (RAGE) during repeated infestations by the tick Haemaphysalis longicornis using RAGE-/- mice. In primary infestation, a large blood pool developed, which was flooded with numerous RBCs, especially during the rapid feeding phase of the tick both in wild-type (wt) and RAGE-/- mice. Very few inflammatory cells were detected around the zones of haemorrhage in the primary infestations. However, the number of inflammatory cells gradually increased in the subsequent tick infestations, and during the third infestations, the number of inflammatory cells reached to the highest level (350.3 ± 16.8 cells/focus). The site of attachment was totally occupied by the inflammatory cells in wt mice, whereas very few cells were detected at the ticks' biting sites in RAGE-/- mice. RAGE was highly expressed during the third infestation in wt mice. In the third infestation, infiltration of CD44+ lymphocytes, eosinophils and expression of S100A8 and S100B significantly increased at the biting sites of ticks in wt, but not in RAGE-/- mice. In addition, peripheral eosinophil counts significantly increased in wt but not in RAGE-/- mice. Taken together, our study revealed that RAGE-mediated inflammation and eosinophils played crucial roles in the tick-induced inflammatory reactions.

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples.

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.

RNA activation in ticks.

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3'- untranslated region (3'-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3'-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

Food- and vector-borne parasitic zoonoses: Global burden and impacts.

Around 25% of the global population suffer from one or more parasitic infections, of which food- and vector-borne parasitic zoonotic diseases are a major concern. Additionally, zoonoses and communicable diseases, common to man and animals, are drawing increased attention worldwide. Significant changes in climatic conditions, cropping pattern, demography, food habits, increasing international travel, marketing and trade, deforestation, and urbanization play vital roles in the emergence and re-emergence of parasitic zoonoses. Although it is likely to be underestimated, the collective burden of food- and vector-borne parasitic diseases accounts for ∼60 million disability-adjusted life years (DALYs). Out of 20 neglected tropical diseases (NTDs) listed by the World Health Organization (WHO) and the Centres for Disease Control and Prevention (CDC), 13 diseases are of parasitic origin. There are about 200 zoonotic diseases of which the WHO listed eight as neglected zoonotic diseases (NZDs) in the year 2013. Out of these eight NZDs, four diseases, namely cysticercosis, hydatidosis, leishmaniasis, and trypanosomiasis, are caused by parasites. In this review, we discuss the global burden and impacts of food- and vector-borne zoonotic parasitic diseases.

Mammalian and Avian Larval Schistosomatids in Bangladesh: Molecular Characterization, Epidemiology, Molluscan Vectors, and Occurrence of Human Cercarial Dermatitis.

Schistosomiasis is a neglected tropical disease (NTD) caused by blood flukes (Schistosoma spp.). Schistosomatids affect a wide array of vertebrate hosts, including humans. In the present study, multiple species of schistosomatids were identified by isolating schistosomatid cercariae (SC) from naturally infected snails. We also described different biotic and abiotic factors influencing SC infections in snails and reported human cercarial dermatitis (HCD) for the first time in Bangladesh. A total of 22,012 snails of seven species: Lymnaea auricularia, L. luteola, Indoplanorbis exustus, Physa acuta, Viviparus bengalensis, Brotia spp., and Thiara spp., were collected and examined. Among these snails, 581 (2.6%) belonging to five species: L. luteola, L. auricularia, P. acuta, I. exustus, and V. bengalensis, were infected with SC. The rate of infection was the highest for L. luteola (11.1%), followed by L. auricularia (5.3%), and was the lowest for V. bengalensis (0.4%). Prevalence in snails was the highest in September (16.8%), followed by October (9.5%) and November (8.8%), and was the lowest in colder months, such as January (1.8%) and February (2.1%). Infections with schistosomatids were more common in larger snails and snails collected from sunny areas. We confirmed the presence of Schistosoma indicum, S. incognitum, S. nasale, S. spindale, and Trichobilharzia szidati by PCR and sequencing. Through a questionnaire survey, we detected HCD in 214 (53.5%) individuals, and the infection rate was almost equally distributed across all professions. Collectively, the present results suggest that lymnaeid snails are the main vector for Schistosoma spp. prevalent in Bangladesh, and schistosomatids with zoonotic potential are also prevalent.

Relationships between footedness and aging on postural control: Evidence from the Yakumo study.

ABSTRACTThe effects of footedness and aging on postural maintenance function were examined in this study. Using the postural maintenance task, the trajectories of the centre of pressure (COP) were measured with a stabilometer in the lateral and posterior/anterior directions. One hundred and ninety-three right-footers (112 middle-aged: 40-64 years old and 81 old-aged: 65-80 years old) and thirty-two left-footers (14 middle-aged and 18 old-aged) among healthy community members participated in this study. Using the ratio of the COP deviation area sizes for the open- vs. closed-eye conditions as an index, the ratios in the lateral and the posterior/anterior directions were compared in relation to the footedness and the age groups. The results indicated that the left-footers of the older adults group showed a larger COP deviation ratio than that of the middle-aged group, and the left-footers showed a larger deviation ratio in the lateral direction than in the posterior-anterior direction. These findings suggest a weak postural maintenance function of the older adults left footers. As most left-footers are left-handed, health promotion staff should notice that older adults who are left-handers are at a higher risk of falling.

Ambivalent Roles of Oxidative Stress in Triangular Relationships among Arthropod Vectors, Pathogens and Hosts.

Blood-feeding arthropods, particularly ticks and mosquitoes are considered the most important vectors of arthropod-borne diseases affecting humans and animals. While feeding on blood meals, arthropods are exposed to high levels of reactive oxygen species (ROS) since heme and other blood components can induce oxidative stress. Different ROS have important roles in interactions among the pathogens, vectors, and hosts. ROS influence various metabolic processes of the arthropods and some have detrimental effects. In this review, we investigate the various roles of ROS in these arthropods, including their innate immunity and the homeostasis of their microbiomes, that is, how ROS are utilized to maintain the balance between the natural microbiota and potential pathogens. We elucidate the mechanism of how ROS are utilized to fight off invading pathogens and how the arthropod-borne pathogens use the arthropods' antioxidant mechanism to defend against these ROS attacks and their possible impact on their vector potentials or their ability to acquire and transmit pathogens. In addition, we describe the possible roles of ROS in chemical insecticide/acaricide activity and/or in the development of resistance. Overall, this underscores the importance of the antioxidant system as a potential target for the control of arthropod and arthropod-borne pathogens.

Research Note: Genetic analysis, pathology, and vectors of echinostomiasis, a zoonotic helminth infection in chickens in Bangladesh.

Echinostomes (Trematoda: Echinostomatidae) are food-borne zoonotic flatworms that affect birds, animals and humans, and has been classified as neglected tropical diseases (NTDs) by the World Health Organization (WHO), which cause severe enteritis in poultry and hamper production. Here, we confirmed the species of echinostomes affecting chickens in Bangladesh along with their genetic analyses, pathology and vectors. We isolated and identified adult worms from chickens, cercariae from fresh water snails and metacerariae (MC) from some wild fishes. We recovered Echinostoma revolutum (10.3%) and Hypoderaeum conoideum (6.0%) from chickens. Zoonotic E. revolutum was confirmed by amplifying nad1 gene and subsequent sequencing. Several mutations were detected in nad1 gene and our isolates belonged to the Euro-Asian clade. We observed thickening of mucosal layer, hyperplasia of goblet cells, infiltration of eosinophils, lymphocytes and must cells in the infected intestine. About 5.3% snails were infected and the highest percentage of infection was found in Lymnaea luteola (12.1%). Echinostome infection in snails was the highest in November (9.6%) and lowest in February (3.1%) in Bangladesh. MC of echinostomes were identified from blue panchax (Aplocheilus panchax) and tank goby (Glossogobius giuris). In conclusion, echinostomiasis is a notable big problem in indigenous chickens in Bangladesh and people, especially, villagers are at risk.

Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection.

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite's mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite's life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.

Data from expressed sequence tags from the organs and embryos of parthenogenetic Haemaphysalis longicornis.

OBJECTIVES: Haemaphysalis longicornis is the most important tick species in Japan and has a wide range of vector capacity. Due to its veterinary and medical importance, this tick species has been used as a model for tick/vector biological studies. To identify the key molecules associated with physiological processes during blood feeding and embryogenesis, full-length cDNA libraries were constructed using the fat body, hemocytes-containing hemolymph, midgut, ovary and salivary glands of fed females and embryos of the laboratory colony of parthenogenetic H. longicornis. The sequences of cDNA from the salivary glands had been already released. However, the related information is still poor, and the other expressed sequence tags have not yet been deposited. DATA DESCRIPTION: A total of 39,113 expressed sequence tags were obtained and deposited at the DNA DataBank of Japan. There were 7745 sequences from embryos, 7385 from the fat body, 8303 from the hemolymph including hemocytes, 7385 from the midgut, and 8295 from the ovary. The data, including expressed sequence tags from the salivary glands was summarized into Microsoft Excel files. Sharing this data resource with the tick research community will be valuable for the identification of novel genes and advance the progress of tick research.

The antiviral immunity of ticks against transmitted viral pathogens.

Ticks, being obligate hematophagous arthropods, are exposed to various blood-borne pathogens, including arboviruses. Consequently, their feeding behavior can readily transmit economically important viral pathogens to humans and animals. With this tightly knit vector and pathogen interaction, the replication and transmission of tick-borne viruses (TBVs) must be highly regulated by their respective tick vectors to avoid any adverse effect on the ticks' biological development and viability. Knowledge about the tick-virus interface, although gaining relevant advances in recent years, is advancing at a slower pace than the scientific developments related to mosquito-virus interactions. The unique and complicated feeding behavior of ticks, compared to that of other blood-feeding arthropods, also limits the studies that would further elaborate the antiviral immunity of ticks against TBVs. Hence, knowledge of molecular and cellular immune mechanisms at the tick-virus interface, will further elucidate the successful viral replication of TBVs in ticks and their effective transmission to human and animal hosts.

Prevalence of Trichinella T9 in Japanese black bears (Ursus thibetanus japonicus) in Iwate prefecture, Japan.

Trichinellosis is a meat-borne zoonotic disease caused by nine Trichinella speices and three unclassified genotypes. In Japan, four domestic outbreaks of human trichinellosis are reported sporadically and were associated with the consumption of wild bear meat. This study examined Trichinella prevalence and its species in black bears, Ursus thibetanus japonicus in Iwate prefecture, Japan. Trichinella T9 larvae identified molecularly were first detected in 1.4% (2/144) of the masseters of black bears examined, and their densities were low (1 and 0.3 larvae /g muscle, respectively). Two cytochrome C oxidase I (COI) haplotypes (sequences) of Trichinella T9 were found in distinct bear populations, suggesting that Trichinella T9 populations isolated genetically by bear populations would occur in Japan.

Natural nodular worm infection in goats induces eosinophil extracellular DNA trap (EET) formation.

Extracellular DNA Trap (ET) formation by granulocyte is a strong innate immune machinery that plays crucial roles in trapping and killing of pathogens. Here, we show Eosinophil Extracellular DNA Trap (EET) formation in goats naturally infected with nodular worms (Oesophagostomum columbianum, Strongyloidae: Nematoda). By a slaughterhouse based survey, we found that 60% goats were infected with nodular worms. We detected numerous, hard and pale yellow to dark black nodules of variable sizes (0.25-2 cm) in the large intestine and the number of nodules were significantly (p < .05) higher in the cecum (21.7 ± 17.9) than in the colon (10.1 ± 9.9). Histologically, pink colored circumscribed caseous mass was surrounded by a dense zone of infiltration and fibrous proliferation along with massive infiltration of eosinophils in and around the necrotic mass. DAPI staining revealed huge accumulation of extracellular DNA, which formed wide ridge like structure surrounding the necrotic zone. Massive release of eosinophils cationic proteins (ECP), a helmintho-toxic substance, was found into the lesions. Collectively, our results suggest that nodular worm infection induces EETosis and ECP release, and is one of the major parasitic problem affecting Black Bengal goats that causes distortion of normal architecture of the gut wall.

Relation between cognitive and cerebello-thalamo-cortical functions in healthy elderly people: Evidence from the Yakumo Study.

Relations between cognitive and cerebello-thalamo-cortical functions in healthy elderly people (65-75 years old) were examined by longitudinal behavioral data. Based on the individually calculated cognitive decline ratio in D-CAT (digit cancelation test) and in LMT (Logical Memory Test) during the period of 11 years, participants were classified into the Decline and the Maintain groups and group differences in the postural tremor measures (Quotient of Romberg) were compared. Significant group differences were shown in the postural tremor measure in D-CAT that reflects prefrontal function, but it was not the case in LMT. These results strengthened our previous findings that suggest a strong relation between the cerebello-thalamo-cortical function and the prefrontal cortex function using behavioral measures. Findings provide evidence that to strengthen postural function such as physical exercise is effective for slowing cognitive decline with age.

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.

Characterization of the RAGE-binding protein, Strongyloides venestatin, produced by the silkworm-baculovirus expression system.

The receptor for advanced glycation end products (RAGE) recognizes Ca++-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca++-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca++ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca++-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes.

Characterization of an iron-inducible Haemaphysalis longicornis tick-derived promoter in an Ixodes scapularis-derived tick cell line (ISE6).

BACKGROUND: Ticks are important vectors of disease-causing pathogens. With the rise of resistance to chemical acaricides, alternative methods in tick control are warranted. Gene manipulation has been successful in controlling mosquitoes and mosquito-borne diseases and is now looked upon as a candidate method to control ticks and tick-borne pathogens. Our previous study has identified the actin and ferritin promoter regions in the Haemaphysalis longicornis tick. RESULTS: Here, the ferritin-derived promoter from the H. longicornis tick was characterized in silico, and the core promoter sequences and some of its important components were identified. Several truncations of the promoter region were created and inserted to a reporter plasmid to determine the important components for its activity. The activities of the truncated promoters on the Ixodes scapularis tick cell line (ISE6) were measured via a dual luciferase assay using experimental and control reporter genes. To induce the promoter's activity, transfected ISE6 cells were exposed to ferrous sulfate. The 639 nucleotides truncated promoter showed the highest activity on ISE6 cells when exposed to 1 mM ferrous sulfate. CONCLUSION: In this study, we characterized an iron-inducible tick promoter that could be a valuable tool in the development of a gene-manipulation system to control ticks and tick-borne pathogens.

Associations of serum microRNA-20a, -27a, and -103a with cognitive function in a Japanese population: The Yakumo study.

OBJECTIVES: MicroRNAs (miRNAs) dysregulate gene expression by binding to target messenger RNAs, and play an important role in the pathogenesis of various diseases, including cancers, cardiovascular diseases and diabetes. Circulating miRNAs have increasingly been recognized as biomarkers for detecting and diagnosing those diseases. Few studies have investigated the association of circulating miRNA with the early stages of cognitive impairment, such as mild cognitive impairment, in the general population. The purpose of this study was to examine the association between cognitive function and several serum miRNAs levels related to amyloid precursor protein (APP) proteolysis in a Japanese general population who had never been diagnosed with dementia. METHODS: We conducted a cross-sectional study of 337 Japanese subjects (144 men, 193 women) who attended a health examination. The short form of the Mini-Mental State Examination (SMMSE) was used to assess cognitive function. Serum levels of 6 miRNAs (let-7d, miR-17, miR-20a, miR-27a, miR-34a, miR-103a) were measured by quantitative real-time polymerase chain reaction. RESULTS: Multivariable-adjusted odds ratios (ORs) for lower SMMSE score (SMMSE score < 28) were significantly increased in the lowest tertile of serum miR-20a (OR, 2.08; 95% confidence interval (CI), 1.09-4.04) and miR-103a (OR, 1.91; 95%CI, 1.00-3.69) compared to the highest tertile. Moreover, serum levels of miR-20a, -27a, and -103a were linearly and positively associated with SMMSE scores after adjustment for confounding factors. CONCLUSION: Low serum levels of miR-20a, -27a, and -103a are independently associated with cognitive impairment.

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella.

Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.