Coinfection of a host by more than 1 parasite is more common than single infection in wild environments and can have differing impacts, although coinfections have relatively rarely been quantified. Host immune responses to coinfection can contribute to infection costs but are often harder to predict than those associated with single infection, due to the influence of within-host parasite–parasite interactions on infection virulence. To first quantify coinfection in a common bird species, and then to test for immune-related impacts of coinfection, we investigated the prevalence and immune response to avian haemosporidian (genera: Plasmodium, Haemoproteus and Leucocytozoon) coinfection in wild blackbirds. Coinfection status was diagnosed using a 1-step multiplex polymerase chain reaction, immune response was quantified through white blood cell counts and heterophil: lymphocyte ratios, and parasitaemia was quantified for each infected sample. We detected high rates of haemosporidian infection and coinfection, although neither impacted immune activity, despite a significantly higher parasitaemia in individuals experiencing double vs single infection. This suggests that immune-related costs of haemosporidian single and coinfection are low in this system. This could be due to long-term host–parasite coevolution, which has decreased infection virulence, or a consequence of reduced costs associated with chronic infections compared to acute infections. Alternatively, our results may obscure immune-related costs associated with specific combinations of coinfecting haemosporidian genera, species or lineages. Future research should investigate interactions that occur between haemosporidian parasites within hosts, as well as the ways in which these interactions and resulting impacts may vary depending on parasite identity.

Different agencies have emphasized the need to evaluate current serological methods for screening patients with suspected urogenital schistosomiasis. However, there is still a lack of evidence regarding the most appropriate methods for this purpose. Here we assessed the diagnostic efficacy of a newly developed serological technique that utilizes the recombinant protein Sh-TSP-2, applied to the urine and serum of migrants suspected of having urogenital schistosomiasis. The sensitivity, specificity, positive and negative predictive values of an in-house enzyme-linked immunosorbent assay (ELISA) using the recombinant protein Sh-TSP-2 were analysed and compared with other commercial serological methods. Due to the limitations of microscopy as a perfect reference method, a latent class analysis (LCA) and composite reference standard (CRS) approach was used to determine the sensitivity and specificity of each test. According to the LCA model, the commercial tests NovaLisa® and immunochromatography test (ICT) immunoglobulin G–immunoglobulin M (IgG–IgM) presented the highest sensitivity (100%), whereas the Sh-TSP-2 serum ELISA test had 79.2%. The Sh-TSP-2 urine and serum ELISA tests had the highest specificities among the serological methods (87.5 and 75%, respectively). CRS modelling showed that the ICT IgG–IgM, NovaLisa® and Sh-TSP-2 serum tests led in sensitivity at 97.1, 88.6 and 71.4%, respectively, with all tests except that the ICT IgG–IgM test having a specificity >90%. Sh-TSP-2 has been validated as a screening tool for patients suspected of having urogenital schistosomiasis. Although commercial serological tests have shown higher sensitivities, Sh-TSP-2 could be valuable for confirming results from tests with lower specificity. Nevertheless, further studies with larger patient cohorts are necessary to fully verify its potential.

The aim was to assess the efficacy of ivermectin vs moxidectin for treating Strongyloides stercoralis infection. Ovid MEDLINE, Embase and Web of Science databases were searched for studies comparing ivermectin and moxidectin from inception to February 2024. The outcomes: elimination of infection or parasitological cure, mortality and serious adverse events. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) for dichotomous data. Heterogeneity was assessed using Chi2 test for statistical heterogeneity and results of the I2 statistic. Two trials met the inclusion criteria that included 821 adult participants. Both studies were conducted in southeast Asia (Cambodia and Laos). Neither trial included immunocompromised patients. The mean age of the participants ranged from 40 to 45 years old, with a similar distribution of males and females. For all participants, S. stercoralis infection was confirmed by Baermann method. The evidence was moderate for parasitological cure rate. Certainty was downgraded by 1 level because of imprecision. Moxidectin was not inferior to ivermectin: OR 0.67, 95% CI 0.36–1.25 (P = 0.21), I2 = 0%, 821 participants. No deaths were reported in either trial. One trial reported mild adverse events. In total, 153/726 (21%) participants had an adverse event. The most reported symptoms were abdominal pain and headache. There is evidence for moderate quality that moxidectin is non-inferior to, and as safe as ivermectin; however, more high-quality and well-designed trials are needed. For patients with some underlying immunosuppressive disorder, or in patients who are very young or very old, current data are insufficient to be recommended.

The nutria was introduced to Europe from South America and kept for the fur industry. This semiaquatic rodent became a well-established species in the Czech Republic; however, it still poses a significant threat to the native fauna, not only as a natural competitor but also as a vector of non-indigenous parasites. Our research aimed to investigate the diversity of endoparasitic helminths in nutria, with a particular focus on assessing the risk posed by helminth species with zoonotic potential. A total of 46 nutria cadavers were collected at 8 locations in the Morava River basin and examined using standard parasitological post-mortem procedures. Additionally, coprological and molecular methods were used to identify the parasites. The presence of 6 helminth species was revealed. The highest prevalence was observed for Strongyloides myopotami (78.3%) and Trichuris myocastoris (37.0%), both of which are host-specific nematodes of nutria. Only 2 trematode taxa were recorded (Echinostoma sp. and a representative of the family Psilostomidae). The presence of alveolar hydatid cysts of Echinococcus multilocularis in the livers of 5 nutria specimens was also recorded. Herein, we provide novel molecular data for each parasite species collected, which is valuable for future phylogenetic analyses. Our findings also demonstrate that nutria in the Czech Republic serve as a carrier of helminths with zoonotic potential, particularly E. multilocularis and S. myopotami. Although the nutria is a relatively new species in local fauna, its synanthropic behaviour raises concerns about potential threats to human health, underscoring the importance of exercising caution when handling these animals.

Gastrointestinal infections constitute a significant global health concern, particularly in tropical and subtropical regions, caused by various pathogens. Among these, Cryptosporidium spp. and Giardia duodenalis are noteworthy due to their zoonotic potential. In Algeria, molecular epidemiological data on cryptosporidiosis and giardiasis are limited. To fill this gap, the present study aimed to examine the transmission dynamics of Cryptosporidium spp., and Giardia duodenalis in various households. A total of 216 samples were collected from the rural Guelma and Souk Ahras provinces, located in the eastern part of Algeria. These included human and animal faeces, as well as water and soil samples. DNA was extracted, followed by nested PCR targeting the SSU rRNA gene to detect Cryptosporidium spp., while the gp60 gene was amplified for subtyping. Detection of G. duodenalis was performed by qPCR targeting the SSU rRNA gene, followed by amplification of tpi, bg and gdh genes for genotyping and subtyping. Several Cryptosporidium species, including C. bovis, C. ryanae, C. andersoni and C. parvum, were identified in human, animal and environmental samples. The zoonotic C. parvum subtype IIaA17G2R1 was detected in human, animal and soil samples. Giardia duodenalis assemblage B was detected in a human sample, while assemblage E was found in cattle and sheep. The current investigation underscores the importance of the One Health approach in addressing issues related to intestinal parasites, highlighting the need for improved surveillance and control measures in rural settings.

Elevated levels of IL-6 in plasma are associated with the severity of visceral leishmaniasis (VL). The clinical manifestations of VL vary among patients, influenced by host factors and the virulence of the Leishmania infantum parasite. Considering that severe VL may result from an exaggerated inflammatory response, this study investigated whether IL-6 could serve as a biomarker to identify pro-inflammatory virulence factors. We conducted a genome-wide association study (GWAS) analysis on L. infantum isolates from patients with VL, whose IL-6 concentrations were measured. The analysis revealed that the relationship between IL-6 levels and clinical outcomes (survival vs mortality) had an area under the curve (AUC) of 0.67 (95% CI 0.52–0.81). A cut-off of 391.7 pg mL−1 for IL-6 was established to conduct a logistic regression analysis. We identified 10 029 single nucleotide variants (SNVs) across 62 genomes, resulting in 6,948 SNVs after filtering, of which 6,341 are located in protein-coding regions. The association analysis with PLINK identified 722 variants, of which 35 showed significant associations, with odds ratios ≥3.3, primarily in coding regions. These findings demonstrate that IL-6 levels tended to be associated with the fatal outcome of VL and highlight 35 novel genetic variants that could serve as potential biomarkers for prognosis. Further research into the biological role of these variants may lead to new therapeutic targets and improve the clinical management of VL, especially in identifying high-risk patients.

Identifying factors that drive among-individual variation in mixed parasitic infections is fundamental to understanding the ecology and evolution of host–parasite interactions. However, a lack of non-invasive diagnostic tools to quantify mixed infections has restricted their investigation for host populations in the wild. This study applied DNA metabarcoding on parasite larvae cultured from faecal samples to characterize mixed strongyle infections of 320 feral horses on Sable Island, Nova Scotia, Canada, in 2014 to test for the influence of host (age, sex and reproductive/social status) and environmental (location, local density and social group membership) factors on variation. Twenty-five strongyle species were identified, with individual infections ranging from 3 to 18 species with a mean richness (±1 s.d.) of 10.8 ± 3.1. Strongyle eggs shed in faeces were dominated by small strongyle (cyathostomins) species in young individuals, transitioning to large strongyles (Strongylus spp.) in adults. Egg counts were highest in young individuals and in the west or centre of the island for most species. Individuals in the same social group had similar parasite communities, supporting the hypothesis that shared environment may drive parasite assemblages. Other factors such as local horse density, sex, date and reproductive/social status had minimal impacts on infection patterns. This study demonstrates that mixed infections can be dynamic across host ontogeny and space and emphasizes the need to consider species-specific infection patterns when investigating mixed infections.

The migration of Mongolian gazelles (Procapra gutturosa) poses a potential risk of outbreak for zoonotic intestinal protozoan parasite infections. This study aims to investigate the infection status of zoonotic intestinal protozoan parasites in these migratory Mongolian gazelles. We collected 120 fecal samples from Mongolian gazelles during their migration from Mongolia to China in December 2023. These samples were analysed using amplification and sequencing of partial SSU rRNA genes to detect the 4 presence of zoonotic intestinal protozoan parasites and characterize their genotypes. Our analysis revealed the presence of several zoonotic intestinal protozoan parasites in the sampled Mongolian gazelles. Cryptosporidium spp. was detected in 14.17% (17/120) of the samples, followed by Cystoisospora belli in 13.33% (16/120), Blastocystis sp. in 16.67% (20/120) and Cyclospora cayetanensis in 30.00% (36/120). Moreover, we identified novel host-adapted genotypes of Cryptosporidium spp. and C. belli, as well as the presence of ST2 and ST13 Blastocystis sp. subtypes, while distinct genotypes were found in Blastocystis sp. and C. cayetanensis. This study revealed the status of 4 prevalent zoonotic intestinal protozoan parasite infections in Mongolian gazelles and provided crucial insights into their characteristics. The prevalence of these parasites in the population highlights the potential risk of cross-border transmission of infectious diseases associated with long-distance migration. Furthermore, the identification of novel genotypes contributes to our understanding of the genetic diversity and adaptation of these parasites. These findings can inform the development of protective measures to mitigate the impact of these infections on the health and survival of Mongolian gazelles.

The functional diversity structure of a community can be represented as a combination of three additive components (species dominance D, functional redundancy R, and functional diversity Q) (DRQ approach in which different facets of functional differences between species are considered simultaneously). We applied this concept to assemblages of fleas and gamasid mites parasitic on small mammals at continental (across regions of the Palearctic) and regional (across sampling sites in Slovakia) scales and asked: What are the relative effects of host species, biome/habitat type, and geographic locality on the DRQ composition of a parasite assemblage? At the continental scale, regions were partitioned according to predominant biome or geographic position in a continental section. At the regional scale, sampling sites were partitioned according to habitat type or geographic locality. We tested for differences in the functional diversity structure (measured as the DRQ composition) of an ectoparasite assemblage (a) within a host species between biomes/habitat types or continental sections/localities and (b) between host species within a biome/habitat type or a continental section/locality. At both scales, the functional diversity structure of both flea and mite assemblages differed mainly between host species within a biome/habitat or geographic regions/locations, whereas differences in the DRQ composition between biomes/habitats or geographic regions/locations were only detected in a few host species. We compare our results with the results of earlier studies and conclude that the DRQ approach has an advantage over a single diversity metric and allows a better understanding of spatial variation in different facets of ectoparasite diversity.

Paleoparasitological studies have made important contributions to our understanding of the past epidemiology of parasites, infection in past populations and lifestyle in the past. In some cases, these ancient parasites can also provide evidence for long distance travel or migration of people in the past. Three sediment samples from a 15th–16th c. CE latrine from the Spanish nation house in Bruges, Belgium were analysed for preserved helminth eggs using microscopy. Bruges was a major trading centre in medieval Europe, thus it was home to a large merchant population with extensive trading networks. Paleoparasitological analysis revealed a preserved parasite egg from Schistosoma mansoni, which causes intestinal schistosomiasis. Roundworm, whipworm, liver fluke and Taenia tapeworm eggs were also found in the latrine which is consistent with parasites previously found in the local population in the medieval period. These new data provide direct evidence for the movement of S. mansoni outside of its endemic area. Today the vast majority of S. mansoni infections occur in Sub-Saharan Africa, with additional endemic areas in the Arabian peninsula and South America. The introduction of S. mansoni into South America is proposed to have occurred relatively recently in human history, as the result of forced movement of people from Africa to the Americas with the Atlantic slave trade. Thus, this infection may have occurred in a merchant who acquired the parasite during trade voyages to Africa or in an individual living in Africa who migrated to Bruges.

Echinococcosis is a parasitic invasion caused by a cestode of the genus Echinococcus. Kyrgyzstan is a country in Central Asia known for an extremely high incidence of echinococcosis. A total of 10 093 subjects were screened in the Osh, Naryn and Batken regions of Kyrgyzstan in 2015–2017 by ultrasound and questioned for potential risk factors. Cystic echinococcosis (CE) prevalence (combined newly diagnosed and post-surgery cases) ranged between 0.2 and 25.2% across the study regions. Typical factors, such as dog or livestock ownership, weakly affected CE risk (odds ratio [OR] = 1.18–1.83). Use of water from a well and owning a cat had a greater effect on CE risk (OR = 2.02–2.28). The risk factors of CE were highly dissimilar among the study regions, with patterns not always compatible with classical biohelminthosis transmission routes (no risk from livestock in certain areas, significant risk from using well water, owning cats). Therefore, the CE epidemic in Kyrgyzstan is not holistic in terms of potential mechanisms and risk factors, and certain areas can greatly benefit from preventive measures that will have limited efficiency elsewhere.

Galba truncatula is one of the most distributed intermediate hosts of Fasciola hepatica across Europe, North Africa and South America. Therefore, understanding the environmental preferences of this species is vital for developing control strategies for fascioliasis and other trematodes such as Calicophoron daubneyi. This systematic literature review evaluates the current understanding of the snail's environmental preferences to identify factors which might aid control and areas where further research is needed. Searches were conducted using Google Scholar and PubMed and included papers published up to August 2023. After filtration, 198 papers with data from 64 countries were evaluated, and data regarding habitat type and habitat pH were noted, along with any other information pertaining to the snail's environmental preferences. The results show that G. truncatula can survive in a diverse range of climates and habitats, generally favours shallow slow-moving water or moist bare mud surfaces, temperatures between 10 and 25°C and was found in habitats with a water pH ranging from 5.0 to 9.4. However, there is limited understanding of the impact of several factors, such as the true optimum pH and temperature preferences within the respective tolerance limits or the reason for the snail's apparent aversion to peatland. Further research is needed to clarify the impact of biotic and abiotic factors on the snail to create robust risk assessments of fluke infection and assess opportunities for environmental control strategies, and for predicting how the snail and fluke transmission may be impacted by climate change.

Echinococcosis lacks sensitive serological diagnostic tools. The echinococcosis-specific antigens Eg95, AgB8/1 and the Em18 gene sequences were fused and expressed as the novel recombinant antigens rAgB8/1-Em18-Eg95 (T3) and rEm18-Eg95 (T2), used for the diagnosis of hydatid disease, prepared into an enzyme-linked immunosorbent reaction (ELISA) kit, and evaluated for their serological diagnostic value. The relative molecular weight of the T3 protein was 88.1 kDa, the purified concentration was 1.5 mg mL−1, and the purity was 80%. The relative molecular weight of T2 protein was 79.9 kDa, the total protein concentration was 0.5 mg mL−1, and the purity was less than 50%. The overall coincidence rate of T2 protein was low, and it was impossible to distinguish between negative and positive sera. The T3 antigen was coated at 1.0 μg mL−1, the cutoff value was 0.5271, and the serum dilution ratio was 1:400. A T3 ELISA kits (96 tests) was constructed to detect the serum of 272 clinically and pathologically confirmed cases. The sensitivity of T3 was 93.8%, and the specificity was 83.3%. The parasite cross-reaction was 30%. Satisfactorily, the Pearson correlation coefficient between the T3 OD value and lesion diameter was 0.707, showing a strong correlation. T3 exhibits better antigenicity than T2, and the prepared T3 ELISA diagnostic kits reached the laboratory diagnostic level of a commercial kits. T3 can distinguish human cystic echinococcosis (CE) and alveolar echinococcosis (AE) more significantly and predict the diameter of lesions according to the OD value, which provides practical value for drug or surgical efficacy.

Amphalius spirataenius belongs to Arthropoda, Insecta, Siphonaptera, Ceratophylloidea, Ceratophyllinae, Amphalius. Only 2 species from the subfamily Ceratophyllinae have been sequenced for mitogenomes to date. The genus Amphalius mitogenome research was still blank. The A. spirataenius mitogenome was determined, annotated and analysed for the first time in this study. The 14 825 bp long genome has the typical metazoan of 37 genes with insect ancestral genome arrangement pattern. There was no significant difference in codon usage of 13 protein-coding genes: UUA, UCU, GUU, ACU and GCU were the most frequently used codons. It was found that the reason for codon preference mainly contributed to natural selection base on PR2, ENC-plot and neutrality curve analysis. Evolutionary rate, conserved sites, variable sites and nucleotide diversity analysis indicated that nad6 of A. spirataenius had the fastest evolutionary rate, while cox1 had the slowest evolutionary rate. Phylogenetic trees were reconstructed based on 13 protein-coding genes and 2 rRNA genes datasets using Bayesian inference and maximum likelihood method. The phylogenetic tree supported that both Siphonaptera and Mecoptera were monophyletic, and were sister groups to each other. This study filled gap of the genus Amphalius mitogenome sequences and was of great significance for understanding evolution of the order Siphonaptera.

Natural infection by Trichinella sp. has been reported in humans and more than 150 species of animals, especially carnivorous and omnivorous mammals. Although the presence of Trichinella sp. infection in wild boars (Sus scrofa) has been documented worldwide, limited information is known about Trichinella circulation in farmed wild boars in China. This study intends to investigate the prevalence of Trichinella sp. in farmed wild boars in China. Seven hundred and sixty-one (761) muscle samples from farmed wild boars were collected in Jilin Province of China from 2017 to 2020. The diaphragm muscles were examined by artificial digestion method. The overall prevalence of Trichinella in farmed wild boars was 0.53% [95% confidence interval (CI): 0.51–0.55]. The average parasite loading was 0.076 ± 0.025 larvae per gram (lpg), and the highest burden was 0.21 lpg in a wild boar from Fusong city. Trichinella spiralis was the only species identified by multiplex polymerase chain reaction. The 5S rDNA inter-genic spacer region of Trichinella was amplified and sequenced. The results showed that the obtained sequence (GenBank accession number: OQ725583) shared 100% identity with the T. spiralis HLJ isolate (GenBank accession number: MH289505). Since the consumption of farmed wild boars is expected to increase in the future, these findings highlight the significance of developing exclusive guidelines for the processing of slaughtered farmed wild boar meat in China.

Angiostrongylus cantonensis, the rat lungworm, is a zoonotic parasite mainly of rats which act as definitive hosts. If humans become accidentally infected, the nematode is capable of migrating to the brain causing meningoencephalitis. Intermediate hosts are snails and slugs. Although originating from mainland China, A. cantonensis has now spread to various countries and continents. The precise timing of its departure from mainland China remains uncertain although it is often associated with significant historical events or migratory movements. The exit of A. cantonensis from mainland China is believed to have occurred in a singular event, followed by its divergence into 2 distinct clades: clade I, originating from mainland China, and clade II, representing global spread. Angiostrongylus cantonensis was first identified in continental Europe in 2021, specifically in Valencia, Spain. Illumina genome sequencing of 7 individuals isolated from rats captured in 2 different districts in the city of Valencia was carried out. The complete mitochondrial genome was assembled and compared with published A. cantonensis mitochondrial genomes through Bayesian phylogenetic analysis, both for complete mitochondrial genomes and for the cytochrome c oxidase I gene, given its widespread use for identification of the species. The findings revealed the presence of 2 different A. cantonensis haplotypes in the rats studied in Valencia, both belonging to clade II. In 2 rats both clades were present.

Visceral leishmaniasis (VL) is a tropical disease that can be fatal if acute and untreated. Diagnosis is difficult, the treatment is toxic and prophylactic vaccines do not exist. Leishmania parasites express hundreds of proteins and several of them are relevant for the host's immune system. In this context, in the present study, 10 specific T-cell epitopes from 5 parasite proteins, which were identified by antibodies in VL patients’ sera, were selected and used to construct a gene codifying the new chimeric protein called rCHI. The rCHI vaccine was developed and thoroughly evaluated for its potential effectiveness against Leishmania infantum infection. We used monophosphoryl lipid A (MPLA) and polymeric micelles (Mic) as adjuvant and/or delivery system. The results demonstrated that both rCHI/MPLA and rCHI/Mic significantly stimulate an antileishmanial Th1-type cellular response, with higher production of IFN-γ, TNF-α, IL-12 and nitrite in vaccinated animals, and this response was sustained after challenge. In addition, these mice significantly reduced the parasitism in internal organs and increased the production of IgG2a isotype antibodies. In vivo and in vitro toxicity showed that rCHI is safe for the mammalians, and the recombinant protein also induced in vitro lymphoproliferative response and production of Th1-type cytokines by human cells, which were collected from healthy subjects and treated VL patients. These data suggest rCHI plus MPLA or micelles could be considered as a vaccine candidate against VL.

Brazil has the richest biodiversity of Ergasilidae on Earth, with 76 species in 19 genera. However, several authors suggested that there is still great potential for discoveries, especially regarding genetic data that are still scarce for ergasilids from this region. To expand our knowledge of this taxon, we conducted an integrative study involving morphological (light and scanning electron microscopy) and molecular analyses of ergasilids from fishes sampled in the Pardo River, São Paulo State, Brazil. Two ergasilid species were found, Gamispatulus schizodontis and Rhinergasilus piranhus parasitizing the nostrils of 5 fish species (native and exotic): (i) R. piranhus from Astyanax lacustris; Cyphocharax modestus; Coptodon rendalli; Psalidodon bockmanni; and (ii) G. schizodontis from Serrasalmus maculatus. Additionally, we redescribed R. piranhus herein based on novelties and discrepancies found in the antennules, antennae, body segments and ornamentation of the swimming legs. Novel molecular data of ribosomal (18S and 28S rDNA) and/or mitochondrial (cox 1) genes were provided for both species and the phylogenetic relationships among the ergasilids were tested. Relationships between species/genera are still uncertain in Ergasilidae, but it was possible to verify the formation of 5 well-supported terminal clades – i.e. including a clade formed only by Neotropical species. More molecular data are needed to test this hypothesis, but the clades observed here represent good targets for future taxonomic revisions. The occurrence of R. piranhus specimens in the introduced fish, Co. rendalli can indicate (for the first time) host switching in this parasite species and spillback from native to introduced hosts.

Austropotamobius pallipes complex is an endangered freshwater crayfish species in Europe and the assessment of the health status of its wild populations is essential for conservation purposes. The two microsporidia Astathelohania contejeani and Nosema austropotamobii have been reported to cause in A. pallipes complex a chronic parasitic infection, known as ‘porcelain disease’, which reduces population fitness and leads the host to death. Due to the similar macroscopic signs produced, molecular biology analyses are required to unambiguously distinguish between these microsporidia. Focusing on A. pallipes complex populations located in Northern and Central Italy, the present study provides an evaluation of prevalence and distribution of A. contejeani and N. austropotamobii, and investigates the variables affecting the probability of detecting infected specimens during a survey (e.g. sex, crayfish density, longitude). Microsporidia were identified in 12 populations among the 42 monitored from 2011 to 2017, with an average prevalence of 3.12% for A. contejeani and 3.60% for N. austropotamobii, the latter being reported in a wider area than previously documented (from Lombardy to Liguria Regions). Notably, crayfish co-infected by both microsporidia were also detected in 4 populations. Moreover, it was observed that the probability of detecting a crayfish with a microsporidian infection significantly increased eastwards in the studied area, especially for N. austropotamobii. Our distribution map for microsporidiosis, combined with molecular screening, will be useful for planning breeding and translocation efforts for A. pallipes complex populations.