Migratory animals likely play an important role in the geographic spread of parasites. In fact, a common assumption is that parasites are potentially transmitted by migratory animals at temporary stopover sites along migratory routes, yet very few studies have assessed whether transmission at stopover sites can or does occur. We investigated the potential for a group of vector-transmitted parasites, the avian haemosporidians, to be transmitted during migratory stopover periods at Rushton Woods Preserve in Pennsylvania, USA. Using an analysis of 1454 sampled avian hosts, we found that while a core group of abundant haemosporidians was shared between local breeding birds and passing migrants, the parasite community of migratory birds at Rushton was distinct from that of local breeding birds and showed similarity to a previously sampled boreal forest haemosporidian community. Haemosporidians that were unique to passing migratory birds were associated with sampling sites in North America with cooler summer temperatures than haemosporidians that are transmitted at Rushton, suggesting that the transmission of these parasites may be restricted to high-latitude regions outside of our temperate stopover site. We also found that the abundance of mosquitoes in our study region is offset from that of migratory bird abundance during avian migratory periods, with the peak period of bird migration occurring during periods of low mosquito activity. Collectively, these findings suggest that although abundant haemosporidians are possibly transmitted between local and passing migratory birds, a combination of biotic and abiotic factors may constrain haemosporidian transmission during avian stopover at our study site.

Oncomelania hupensis (O. hupensis), the sole intermediate host of Schistosoma japonicum, greatly influence the prevalence and distribution of schistosomiasis japonica. The distribution area of O. hupensis has remained extensive for numerous years. This study aimed to establish a valid agent-based model of snail density and further explore the environmental conditions suitable for snail breeding. A marshland with O. hupensis was selected as a study site in Dongting Lake Region, and snail surveys were monthly conducted from 2007 to 2016. Combined with the data from historical literature, an agent-based model of snail density was constructed in NetLogo 6.2.0 and validated with the collected survey data. BehaviorSpace was used to identify the optimal ranges of soil temperature, pH, soil water content, and vegetation coverage for snail growth, development and reproduction. An agent-based model of snail density was constructed and showed a strong agreement with the monthly average snail density from the field surveys. As soil temperature increased, the snail density initially rose before declining, reaching its peak at around 21°C. There were similar variation patterns for other environmental factors. The findings from the model suggested that the optimum ranges of soil temperature, pH, soil water content and vegetation coverage were 19°C to 23 °C, 6.4 to 7.6, 42% to 75%, and 70% to 93%, respectively. A valid agent-based model of snail density was constructed, providing more objective information about the optimum ranges of environmental factors for snail growth, development and reproduction.

The challenge of American tegumentary leishmaniasis (ATL) continues in Brazil, presenting a persistent public health issue despite initiatives aimed at public outreach, vector control and health education. To gain a deeper understanding of this disease, a study was conducted in an endemic region located in the northern region of the state of Minas Gerais, Brazil. The study monitored 30 resident patients diagnosed with ATL, using serum samples from 6 healthy individuals as controls. The localized cutaneous form of the disease was found to be predominant, with lesions appearing on various parts of the body and the majority of the affected individuals being male. The study found significantly higher levels of IgG anti-α-Gal antibodies in ATL-infected patients compared to healthy individuals. Treatment of 19 patients with meglumine antimoniate resulted in limited improvement in symptoms for most. Nonetheless, the study found that 12 patients who completed treatment with epithelialization of the lesions showed a significant decrease in IgG anti-α-Gal antibodies, indicating potential applications of this antibody in the diagnosis and monitoring of the disease. The study also identified Leishmania species in 7 analysed patients, revealing 6 cases infected by Leishmania braziliensis and 1 by L. infantum, with a significant difference in the anti-α-Gal responses. The findings of the study emphasize the urgent need for the development of human vaccines and innovative treatment strategies adapted to the diversity of Leishmania species causing cutaneous leishmaniasis and individual patient responses to improve the clinical management of ATL in Brazil and similar endemic regions.

The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the 4 distinctive extended winged-helix (eWH) domains and the coiled-coil motif are present in C82 in these microorganisms. Nevertheless, C82 in trypanosomatids presents certain unique traits, including an exclusive loop within the eWH1 domain. We found that C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. Knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of T. brucei. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.

Most studies concerning parasitic infections in European bison have been performed on free-ranging animals: comparatively little is known about the abomasal nematodes of captive wisents, which are widely used in reintroduction programmes. The aim of the study was to determine the infection level and species composition of abomasal nematodes in captive European bison in enclosures (including zoos) and breeding centres compared to free-ranging individuals. It also includes a morphological analysis of the parasites based on figures and measurement data. Altogether, 11 species of nematodes were detected, with both captive and free-ranging animals demonstrating similar species compositions. Among those, 2 species of blood-sucking nematodes were detected, including Ashworthius sidemi and Haemonchus contortus. Interestingly, A. sidemi was found in almost all free-roaming animals, but only in 1 captive European bison. In addition, H. contortus was predominant in captive animals. The morphological identification was confirmed molecularly for 5 nematode species: A. sidemi, H. contortus, Ostertagia kolchida, O. ostertagi and Spiculopteragia boehmi. The identification was performed using small subunit ribosomal rDNA. The study provides the first available set of specular lengths of the gastric nematodes of European bison, and the first molecular data of O. kolchida and S. boehmi derived from the same host species. Our findings may simplify the morphometrical and molecular identification of Trichostrongylidae species infecting European bison, and can be useful in developing new management strategies for populations of this near-threatened species in Europe.

Cystic echinococcosis (CE), caused by Echinococcus granulosus s.l. is a neglected zoonosis posing a significant public health challenge. Little is known about human CE in Bhutan. This study was conducted to gain an understanding of the burden, distribution, and potential risk factors of CE in Bhutan. From January 2015 to December 2019 data from Jigme Dorji Wangchuck National Referral Hospital (JDWNRH) and 6 other district-level hospitals were reviewed. Descriptive statistics were used to summarize the data. DALYs and Poisson regression models were used to estimate the burden and explore the relationship between cases and possible risk factors. A total of 159 cases were recorded. Most cases (145) were admitted to the surgical ward and 14 cases were referred to India. The average annual incidence was 4.4 cases per 100 000 population. The burden of disease was estimated to be approximately 39 DALYs per year for treatment-seeking cases, or possibly 80 DALYs per year including non-treatment seeking cases. This translates to approximately to 5.2 DALYs and 10.2 per 100 000 per year respectively. The commonest sites of infection were the liver (78%) and lungs (13%). Most cases were treated with surgery (>82%), and more than 47% were admitted to the hospital for >4 days. Policy interventions targeting community engagement, awareness, education, high risk occupational groups, females, and those living in the endemic districts of the central and western regions may yield larger gains. More studies and the institution of a surveillance system can help better guide policy interventions.

Human alveolar echinococcosis is a hard-to-treat and largely untreated parasitic disease with high associated health care costs. The current antiparasitic treatment for alveolar echinococcosis relies exclusively on albendazole, which does not act parasiticidally and can induce severe adverse effects. Alternative, and most importantly, improved treatment options are urgently required. A drug repurposing strategy identified the approved antimalarial pyronaridine as a promising candidate against Echinococcus multilocularis infections. Following a 30-day oral regimen (80 mg kg−1 day−1), pyronaridine achieved an excellent therapeutic outcome in a clinically relevant hepatic alveolar echinococcosis murine model, showing a significant reduction in both metacestode size (72.0%) and counts (85.2%) compared to unmedicated infected mice, which revealed significantly more potent anti-echinococcal potency than albendazole treatment at an equal dose (metacestode size: 42.3%; counts: 4.1%). The strong parasiticidal activity of pyronaridine was further confirmed by the destructive damage to metacestode tissues observed morphologically. In addition, a screening campaign combined with computational similarity searching against an approved drug library led to the identification of pirenzepine, a gastric acid-inhibiting drug, exhibiting potent parasiticidal activity against protoscoleces and in vitro cultured small cysts, which warranted further in vivo investigation as a promising anti-echinococcal lead compound. Pyronaridine has a known drug profile and a long track record of safety, and its repurposing could translate rapidly to clinical use for human patients with alveolar echinococcosis as an alternative or salvage treatment.

Ixodes ricinus, commonly known as the castor bean tick and sheep tick, is a significant vector of various diseases, such as tick-borne encephalitis and Lyme borreliosis. Owing to climate change, the distribution and activity of I. ricinus are expected to increase, leading to an increase in the number of diseases transmitted by this species. Most distribution models and ecological niche models utilize macroclimate datasets such as WorldClim or CHELSA to map the distribution of disease-transmitting ticks. However, microclimatic factors are crucial for the activity and survival of small arthropods. In this study, an ecological niche modelling approach was used to assess the climatic suitability of I. ricinus using both microclimatic and macroclimatic parameters. A Mixed model was built by combining parameters from the Soiltemp (microclimate) and Wordclim (macroclimate) databases, whereas a Macroclimate model was built with the CHELSA dataset. Additionally, future suitabilities were projected via the macroclimate model under the SSP3-7.0 and SSP5-8.5 scenarios. Macroclimate and Mixed models showed similar distributions, confirming the current distribution of I. ricinus. The most important climatic factors were seasonality, annual temperature range, humidity and precipitation. Future projections suggest significant expansion in northern and eastern Europe, with notable declines in southern Europe.

Communities living in African animal trypanosomiasis (AAT) endemic areas of Zambia use several control strategies to protect their livestock from the devastating effects of trypanosomiasis. Several studies have reported the effectiveness of trypanosomiasis control strategies based on retrospective data. In this study, we assessed incidence rates of AAT in cattle (n = 227) using a prospective cohort study comprising 4 treatment groups, i.e., Diminazene aceturate, Isometamidium chloride, Cyfluthrin pour-on and Cypermethrin treated targets. The study was conducted in Mambwe district in Eastern Zambia between February 2019 and March 2020. The endemic prevalence of AAT for each group was determined using ITS-PCR prior to application of treatments. High endemic trypanosome pre-treatment rates were found in all Groups (Diminazene aceturate (61%), Isometamidium chloride (48%), Cyfluthrin pour-on (87%) and Cypermethrin targets (72%)). The overall apparent prevalence for the Mambwe district was 67% (152/227) and true prevalence at 95%CI was 63–71%. Once treatments were implemented, 12 monthly follow-ups were conducted. The average monthly incidence rates without standardization recorded: Diminazene aceturate (67%) Isometamidium chloride (35%), Cyfluthrin pour-on (55%) and Cypermethrin targets (61%). Incidence rates were standardized considering the endemic level of disease for each Group and the average standardized monthly incidence rate in the Diminazene aceturate Group was 7%; the Isometamidium chloride Group −13%; the Cyfluthrin Group −26%; and the Cypermethrin target Group, −17%. All Groups showed a decrease in incidence of AAT over the period of the study with the Cyfluthrin group showing to be the most effective in reducing AAT incidence in cattle.

A new species of Myxobolidae, Henneguya cardii n. sp., is described infecting the European seabass Dicentrarchus labrax, a fish of high commercial value intensively cultivated in southern Europe. Henneguya cardii n. sp. was found in the bulbus arteriosus and spleen with a prevalence of infection of 13.5%. In the heart, it forms irregular whitish plasmodia measuring 1 mm in size. Mature myxospores are broadly obovoid in frontal view and ellipsoidal in lateral view, with 2 equal caudal appendages. Polar capsules are ovoid and symmetric, with 3–4 polar tubule coils. Myxospores measure 10.2 ± 0.6 (8.8–11.6) μm in length, 8.0 ± 0.7 (5.3–8.8) μm in width and 5.6 ± 0.2 (5.1–6.4) μm in thickness. Caudal appendages are 36.6 ± 3.2 (27.4–42.9) μm long. Total spore length is 47.6 ± 3.2 (41.2–53.2) μm. Polar capsules measure 4.0 ± 0.2 (3.4–4.6) by 2.2 ± 0.1 (1.9–2.6) μm. Small subunit ribosomal RNA-based tree topologies position H. cardii n. sp. within a lineage of marine myxobolids that is mostly comprised of other Henneguya species. Host-relatedness is reinforced as the main evolutionary driver for myxobolids, with the positioning of H. cardii n. sp. further suggesting tissue tropism as another important evolutionary driver for marine heart infecting Henneguya. Nonetheless, the inner complexity of this lineage suggests that identification of the evolutionary patterns driving its phylogeny will require discovery of the true diversity of marine myxobolids.

The study aimed to assess the heterogeneity in the distribution of disease awareness, attitudes, and practices related to cystic echinococcosis (CE) in different subgroups and inform health authorities regionally and globally for future evidence-based tailored prevention practices in the region. A cross-sectional study was conducted with 242 participants from Kyrgyz Republic (KR), Issyk-Kul oblast, and utilized survey data to analyse demographics, household information, echinococcosis-related practices, and knowledge. Participants in high-risk environments (HRE) and engaging in high-risk behaviours (HRB) linked to CE contracting were identified. Out of 242 participants, 39% lived in HRE, with 22% engaging in HRB of contracting CE. 13% lived in HRE and engaged in HRB. Only 6% followed all preventive measures, while 56% followed some. 97.5% of participants had heard about CE, but only 6% identified all transmission routes, and 63.4% were unaware of dog contact as a route. Education reduced the odds of being in the highest risk group (HRE&HRB) (OR 0.5, 95% CI 0.23–0.80). The study's findings are alarming, emphasizing factors contributing to regional endemicity. We anticipated a similar pattern in the neighbouring countries, given the shared nomadic customs and historical parallels. Examination of the heterogeneity of disease awareness and practices allows tailored prevention strategies. Urgent prevention programmes focusing on echinococcosis awareness in the KR are crucial to addressing challenges posed by nomadic habits.

Dioctophyme renale, the giant kidney worm, is a nematode related to Trichuris sp and is distributed worldwide. These parasites locate in the kidney of their definitive hosts (mainly belonging to the order Carnivora) and have an indirect life cycle with an annelid as the main intermediate host. Humans are rarely affected, but in those that are, 1 or both kidneys are destroyed. In South America, D. renale is widespread in riparian regions where changing climatic conditions, environmental degradation, and compromised sanitation are increasing the risk of distribution of this parasite, including humans. Here, we provide the descriptions of the genetic diversity of the parasite in the region by analysing 73 adult D. renale samples collected from domestic and wild carnivores. The most common hosts were (Canis lupus familiaris) and maned wolf (Chrysocyon brachyurus Fam. Canidae) among domestic and wild carnivores, respectively. This work shows the descriptions of the genetic diversity of this parasite complementing molecular methods and classical and probabilistic phylogeography. Our results strongly suggest that this parasite has been present on the continent long enough to develop local genetic variants. Also, the phylogenies show transmission between localities and bidirectional transmission between domestic and wild species. We now have new tools to understand the ecological dynamics of this parasite such as molecular markers to study its genetic diversity as well as for identification and reporting in cryptic cases.

A knowledge, attitudes and control practices (KAP)-based study on ticks and tick-borne diseases (TTBD) and resistance development in ticks was conducted in Dhar district of Madhya Pradesh covering 200 livestock owners using a questionnaire. Based on our scoring criteria, results indicated only 25% (19.16–31.60) respondents possessing basic knowledge of TTBDs while 75% (68.40–80.84) respondents were not aware of TBDs. Due to lack of proper awareness of TTBDs, about 1.28 times more respondents (OR 95% CI 0.42–3.86) were having heavy tick infestations in their animals. However, about 36.5% (29.82–43.58) respondents showed a favourable attitude towards the adoption of different tick control practices; consequently, their animals showed low-level infestation. Amongst various feeding systems for animals, a mixed type of feeding system was mostly adopted by 57.5% respondents followed by manger system (37.5%) while grazing was the least adopted method (5%). Results indicated that the grazing animals were 6 times (OR 95% CI 2.93–12.28) more susceptible to ticks and possessed heavy tick infestation. Resistance status of collected tick isolates of Rhipicephalus microplus and Hyalomma anatolicum was assessed and revealed that both tick species were found resistant to deltamethrin. The goals of this study were to assess some of the underlying causes of ticks and TBD in livestock in Dhar district of Madhya Pradesh state using the KAP survey and resistance characterization of ticks.

Mastomys natalensis and M. coucha are commensal rodent species endemic to Africa. A recent taxonomic revision within Mastomys leaves the parasite–host list of M. natalensis questionable and that of M. coucha incomplete. The current study aimed to develop a better understanding of the ectoparasite diversity associated with the 2 distinct but closely related rodent species and to explore the influence of host and habitat type on ectoparasite infestations. Between 2014 and 2020, 590 rodents were trapped in 3 habitat types (village, agriculture and natural) across a wildlife-human/domestic animal interface. In total 48 epifaunistic species (45 ectoparasitic and 3 predatory) represented by 29 genera from 4 taxonomic groups (fleas, lice, mites and ticks) were recorded. Only 50% of the epifauna were shared between the 2 rodent species, with mites the most speciose taxon in both host species. The abundance of epifaunistic individuals, and also those of mites and fleas, were significantly higher on male M. natalensis, while ticks were significantly higher on reproductively active M. natalensis. For both rodent species, infestations by most epifaunistic taxa (on M. natalensis) and some taxa (on M. coucha) were significantly lower in the village as opposed to the less disturbed agricultural and natural habitat types. The study highlights the importance of host life history, even in closely related rodent species, in shaping parasite profiles and a loss of parasite diversity in more extreme anthropogenic habitats.

We report a case of a suspected intestinal myiasis of a child from Southeast Argentina. Diptera larvae were sampled by a physician from the nappy worn by the child and submitted for examination and identification to the Laboratorio de Artrópodos (Universidad Nacional de Mar del Plata). Based on diagnosis of the anterior and posterior spiracles and mouthparts, the larvae were identified as the false stable fly Muscina stabulans (Fallén, 1817) (Diptera: Muscidae). A comparison of diagnostic characters between Argentinean and European third instars of this species is presented. Muscina stabulans is a prevalent species in the district of General Pueyrredón, Buenos Aires province, where the case occurred. Its abundance in the area coincidentally peaked at the time of the infestation. This is the 1st report of M. stabulans as a suspect of intestinal myiasis for the whole of the southern cone of South America (Chile and Argentina).

Schistosomiasis is a neglected tropical disease with significant health implications, particularly among children. A cross-sectional study was conducted among school-aged children (SAC) in Mwanga district, Tanzania, a region known to be co-endemic for S. haematobium and S. mansoni infection and where annual mass drug administration (MDA) has been conducted for 20 years. In total, 576 SAC from 5 schools provided a urine sample for the detection of Schistosoma circulating anodic antigen using the upconverting particle-based lateral flow (UCP-LF CAA) test. Additionally, the potential of the point-of-care circulating cathodic antigen (POC-CCA) and microhaematuria dipstick test as field-applicable diagnostic alternatives for schistosomiasis were assessed and the prevalence outcome compared to UCP-LF CAA. Risk factors associated with schistosomiasis was assessed based on UCP-LF CAA. The UCP-LF CAA test revealed an overall schistosomiasis prevalence of 20.3%, compared to 65.3% based on a combination of POC-CCA and microhaematuria dipstick. No agreement was observed between the combined POC tests and UCP-LF CAA. Factors associated with schistosomiasis included age (5–10 years), involvement in fishing, farming, swimming activities and attending 2 of the 5 primary schools. Our findings suggest a significant progress in infection control in Mwanga district due to annual MDA, although not enough to interrupt transmission. Accurate diagnostics play a crucial role in monitoring intervention measures to effectively combat schistosomiasis.

Cystic and alveolar echinococcosis are considered the second and third most significant foodborne parasitic diseases worldwide. The microscopic eggs excreted in the feces of the definitive host are the only source of contamination for intermediate and dead-end hosts, including humans. However, estimating the respective contribution of the environment, fomites, animals or food in the transmission of Echinococcus eggs is still challenging. Echinococcus granulosus and E. multilocularis seem to have a similar survival capacity regarding temperature under laboratory conditions. In addition, field experiments have reported that the eggs can survive several weeks to years outdoors, with confirmation of the relative susceptibility of Echinococcus eggs to desiccation. Bad weather (such as rain and wind), invertebrates and birds help scatter Echinococcus eggs in the environment and may thus impact human exposure. Contamination of food and the environment by taeniid eggs has been the subject of renewed interest in the past decade. Various matrices from endemic regions have been found to be contaminated by Echinococcus eggs. These include water, soil, vegetables and berries, with heterogeneous rates highlighting the need to acquire more robust data so as to obtain an accurate assessment of the risk of human infection. In this context, it is essential to use efficient methods of detection and to develop methods for evaluating the viability of eggs in the environment and food.

Sex biases in prevalence of disease are often attributed to intrinsic factors, such as physiological differences while a proximate role of extrinsic factors such as behavioural or ecological differences may be more difficult to establish. We combined large-scale screening for the presence and lineage identity of avian malaria (haemosporidian) parasites, in 1234 collared flycatchers (Ficedula albicollis) with life-history information from each bird to establish the location and timing of infection. We found an overall infection rate of 36.2% ± 0.03 (95% CI) with 25 distinct malaria lineages. Interestingly, first-year breeding males and females had similar infection prevalence while females accrued a significantly higher infection rate than males later in life. The sex difference in infection rate was driven by the most abundant Haemoproteus, lineage, hPHSIB1, while the infection rate of Plasmodium lineages was similar in males and females. Furthermore, when infections were assigned to an apparent transmission location, we found that the sex difference in infection rate trend was driven by lineages transmitted in Europe, more specifically by one lineage (the hPHSIB1), while no similar pattern was found in African lineages. We deduce that the observed infection patterns are likely to be caused by differences in breeding behaviour, with incubating females (and nestling individuals of both sexes) being easy targets for the biting insects that are the vectors of avian malaria parasites. Overall, our results are most consistent with ecological factors rather than intrinsic factors underlying the observed sex-biased infection rate of avian malaria in collared flycatchers.

This study aimed to explore extracellular microRNA derived from Echinococcus multilocularis (EM) in the plasma of patients with alveolar echinococcosis (AE) and assess its potential as a diagnostic biomarker. EM-derived miRNAs were identified in plasma samples from 20 AE patients through miRNA sequencing. Three novel miRNA molecules (emu-miR-novel 1, 2 and 3) were predicted through bioinformatic analysis to elucidate their chromosomal locations, secondary structures and precursor forms. Subsequently, plasma samples from 30 AE patients and 30 controls were utilized to establish an assay via stem-loop reverse transcription PCR, optimizing primers, reaction systems, and conditions to assess cross-reactivity and sensitivity. Clinical validation revealed that emu-miR-novel 1 had the highest diagnostic accuracy, with an area under the curve (AUC) of 0.8994, a P value of less than 0.0001, a sensitivity of 83.3%, and a specificity of 86.7%. Statistically significant differences were observed between the groups for emu-miR-novel 1 (P < 0.05), whereas emu-miR-novel 2 and 3 showed AUC values of 0.7922 and 0.6883, with P values of 0.0001 and 0.012, respectively, indicating no significant difference between groups (P > 0.05). Furthermore, the assay showed no cross-reactivity with samples from 18 common viruses, 4 parasitic infections, and miRNAs from AE sequenced from 8 species, confirming its high specificity. Emu-miR-novel 1 exhibited a sensitivity of 1 femtomolar. Emu-miR-novel 1 holds promise as a key diagnostic tool for AE, offering a novel perspective and approach for disease diagnosis.

Coccidiosis is a parasitic disease caused by Eimeria spp., and the emergence of drug resistance has seriously affected the control of the disease. Using RNA-seq, we previously found that phosphoglycerate kinase of Eimeria tenella (EtPGK) was differentially downregulated in diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains compared with drug-sensitive (DS) strain. In this study, we further analysed the characteristics and functions of EtPGK to find the possible mechanism of drug resistance of E. tenella. Quantitative real-time PCR (qRT-PCR) and western blot found that EtPGK was highly expressed in sporulated oocysts, followed by sporozoites and second-generation merozoites of E. tenella. Indirect immunofluorescence localization showed that EtPGK was located mainly in the cytoplasm and on the surface of the parasites. Invasion inhibition assays showed that anti-rEtPGK antibody significantly inhibited the invasion of parasites. Further studies using qRT-PCR and western blot found that the transcription and translation levels of EtPGK were downregulated in both resistant (DZR and MRR) strains compared with the DS strain, and the transcription level correlated negatively with the drug concentration. The enzyme activity assay revealed that EtPGK enzyme activity was decreased in the DZR strain compared with the DS strain. qRT-PCR revealed that the mRNA transcription level of EtPGK was significantly downregulated in the field DZR strain and salinomycin-resistant strain compared with the DS strain. These results suggested that EtPGK has other important roles that are separate and distinct from its function in glycolysis, and it might be involved in the development of drug resistance of E. tenella.