Seasonal Dynamics and Potential Interactions of Haematophagous Abomasal Nematodes in two Chamois populations in the Czech Republic

Seasonal Dynamics and Potential Interactions of Haematophagous Abomasal Nematodes in two Chamois populations in the Czech Republic | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Seasonal Dynamics and Potential Interactions of Haematophagous Abomasal Nematodes in two Chamois populations in the Czech Republic Jan Magdálek, Vojtěch Kasič, Jana Ilgová, Lucie Škorpíková, Jaroslav Vadlejch This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6529385/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Sep, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 17 You are reading this latest preprint version Abstract Background: Pathogenic blood-feeding nematodes, such as Haemonchus contortus and the invasive Ashworthius sidemi , infect a wide range of wild and domestic ruminants. While the spread of A. sidemi among European cervids has been studied, its presence in chamois ( Rupicapra rupicapra ) remains poorly documented. Conversely, H. contortus is known to infect chamois, but previous research has relied mainly on cross-sectional necropsy studies, offering only a limited view of infection dynamics. In this study, we used a longitudinal molecular approach to assess the seasonal occurrence and transmission patterns of H. contortus and A. sidemi in a chamois population from the northern Czech Republic. From January to December 2023, we collected faecal samples at monthly intervals from two localities. Multiplex real-time PCR was subsequently used for the detection and semi-quantification of DNA from both nematode species. Results: Haemonchus contortus DNA was detected in 43.3% of samples, with its presence recorded nearly year-round. Its prevalence and relative abundance peaked in summer and remained high throughout autumn. Ashworthius sidemi was identified in chamois in the Czech Republic for the first time, likely due to recent spillover from red deer ( Cervus elaphus ). However, it was found in only 5% of samples, with its occurrence restricted to late winter and spring. The seasonal disappearance of A. sidemi coincided with the increase in H. contortus abundance, suggesting a possible negative interaction between these species occupying the same ecological niche. Conclusion: Our findings indicate a prolonged transmission window for H. contortus , which may expand further with climate change. In contrast, A. sidemi appears to be an incidental parasite in chamois, and its long-term persistence in this atypical host without continued contact with cervids remains uncertain. These insights, which are rare for wild ruminants, contribute to a better understanding of parasite epidemiology and host-parasite interactions in free-living populations. Chamois Haemonchus contortus Ashworthius sidemi Real-time PCR Prevalence Epidemiology Figures Figure 1 Figure 2 1. Background The chamois Rupicapra rupicapra is distributed to higher altitudes and specific, rocky terrain types; however, the species exhibits remarkable adaptability, allowing it to thrive in a wide range of mountainous environments [ 1 ]. The chamois is considered a non-native species in the Czech Republic (CR) as the presence of two populations of chamois in the country has its origin in introductions that took place at the beginning of the 20th century to enrich the spectrum of the species for trophy hunting. These introductions were repeatedly carried from the alpine regions of Austria and Germany, as well as from zoos and game breeders in Austria and Switzerland [ 2 ]. Analysis of the mitochondrial control region shows that the current chamois populations in the CR, found in the northern part of the country (The Lusatian Mountains Protected Area (PLA) and Jeseníky Mountains, are most genetically related to northern chamois populations in the eastern Alps, specifically in the Ebensee and Mürzsteg regions [ 3 ]. As the mountain chamois is restricted to specific habitats and has low dispersal rates, gene flow is limited, resulting in genetically distinct populations. Introduced populations in Central Europe have shown lower genetic variability than their Alpine counterparts, and apparent consequences of founder effects and bottleneck events [ 4 ], which could potentially increase their susceptibility to pathogens. Chamois host a variety of gastrointestinal parasites, with those infecting the abomasum being the most significant. While typically subclinical [ 5 ], these infections can negatively impact the host's fitness and welfare. A negative correlation between the species richness of abomasal nematodes and both skeletal development and nutritional status of alpine chamois in Italy has been observed [ 6 ]. Among these parasites, Haemonchus contortus is considered the most important for chamois health [ 7 , 8 ]. Although primarily associated with significant losses in small livestock farms worldwide [ 9 ], this blood-sucking nematode has low host specificity and can spread between grazing livestock and wild ruminants, including northern chamois [ 10 ]. This parasite was identified as the direct cause of death in only 1.4% of postmortem-examined chamois in the Slovenian Alps [ 11 ]. However, its broader impact should not be underestimated as subclinical infections may weaken individuals, making them more susceptible to other stressors. In the Italian Alps, H. contortus was reported as a predominant abomasal parasite and suggested as a predisposing factor contributing to a pneumonia-related mass die-off [ 12 ]. In addition, a close relative of the parasite H. contortus from the subfamily Haemonchinae, Ashworthius sidemi , has recently been identified in a hybrid of alpine ( R. rupicapra rupicapra ) and tatra chamois ( R. rupicapra tatrica ) from the Low Tatras (Slovakia) [ 13 ]. The original hosts of this nematode are thought to be the cervids of eastern Asia, but with the translocation of sika deer ( Cervus nippon ), it was inadvertently repeatedly introduced into central and eastern Europe and France, as reviewed by Rehbein et al. [ 14 ]. It has successfully transferred from its original host to various local ruminant species and, in Europe, currently occurs locally with a high prevalence [ 15 ]. In addition, with new hosts, there is likely to be further natural spread of the parasite into new territories [ 14 , 16 – 18 ]. The parasite was first recorded in the former Czechoslovakia in the 1970s at the Lány game reserve [ 19 ], and recent molecular monitoring indicates that it is now most common in red deer ( Cervus elaphus ) [ 20 ]. In that study, the A. sidemi infection was also confirmed in red deer in the locality of chamois population occurrence in the northern part of the CR. Despite the different feeding strategies of red deer and chamois [ 21 ], the diet composition observed in the other chamois locality, the Jeseníky Mountains, was almost identical for both species. Both diets were primarily composed of grasses, with smaller proportions of herbs and woody broad-leaved plants [ 22 ]. This overlap in ecological niches suggests the potential for transmission of the invasive A. sidemi . Gastrointestinal nematode data from chamois have typically been collected through necropsy-based cross-sectional studies, e.g. [ 7 , 12 , 13 ]. While these methods enable precise species identification and infection intensity estimates, they provide only a narrow insight into fluctuating infection status, as sampling is usually limited to the autumn hunting season. Alternatively, coprological analysis offers a non-invasive way to monitor seasonal parasite output [ 23 , 24 ]. However, this method does not allow species-level identification, as most strongylid parasite eggs are morphologically indistinguishable, limiting its ability to track the most pathogenic species. For these reasons, we adopted the real-time PCR method proposed by Reslová et al. [ 25 ], which enables reliable differentiation of the haematophagous nematodes A. sidemi and H. contortus , as well as semi-quantification directly from faecal samples. The main goals of this study were to investigate the occurrence of the invasive species A. sidemi and the important pathogen H. contortus in a northern chamois population from the Lusatian Mountains PLA (CR) and to monitor seasonal variations in egg production of both parasites as well as explore potential interactions between them. 2. Methods 2.1 Study site and animals The chamois population in the northern part of the Czech Republic inhabits three protected areas: the Czech Switzerland National Park, the Lusatian Mountains PLA, and the Elbe Sandstones PLA. To monitor the occurrence and seasonal variation in A. sidemi and H. contortus egg production, we selected two areas with the highest chamois presence: one in the Lusatian Mountains PLA and the other in the Elbe Sandstones PLA. These study sites were approximately seven km apart aerially, separated by fields, urban development, and a traffic road. Based on the Köppen–Geiger climate classification [ 26 ], the study area is characterized as a warm-summer humid continental climate, typical for Central Europe. This climate features clearly defined seasons, with warm but not excessively hot summers, long and cold winters, and generally lower levels of precipitation throughout the year [ 27 ]. The locality A, Studený Vrch, lies adjacent to the Studenec Hill (50°49′56″ N, 14°27′16″ E ) with its peak at an altitude of 737 m. The terrain consists of volcanic stony fields, partly refugial post-glacial basiphilous beech mixed fir-beech forests (alliance Fagion sylvaticae ) and meadows dominated by false oatgrass ( Arrhenatherum elatius ) grazed by chamois during the vegetation period. Based on the census carried out by the local forestry service, the hunting ground covering this site was inhabited by 39 individuals in 2023. From January to April, supplementary feeding of concentrates was carried out, while from March onwards, the movement to meadows and grazing was allowed. From the end of January until approximately mid-March, a continuous snow cover was observed at this site, except for one week at the end of February. Feeding took place on common feeders with red deer, whose numbers were estimated at 78 individuals on the hunting ground. During the grazing season, deer and chamois used the same meadows, but both species grazed mostly in separate parts. Mixed groups were not observed during the daytime. The second monitored site (locality B) was located in the Česká Kamenice hunting ground, specifically on Strážiště Hill (50°48′59″N, 14°22′19″E) at an altitude of 469 m. This area is characterised by generally lower elevation and is dominated by Man-made forests with high prevalence of Picea sp. and vegetation from the Luzulo-Fagetum association. The number of chamois at this site was estimated at 47 individuals by the local forestry service. Supplementary feeding was limited here to the winter season and occurred at feeding stations specially adjusted to be accessible only to chamois without disturbance by the red deer. Simultaneously, grazing was carried out all year round, with mixed groups of chamois and deer observed at this site. The total number of deer on this hunting ground was estimated at 126. 2.2 Sample collection Freshly excreted faecal samples were collected monthly from both sites. The animals were first observed with binoculars outside their flight distance to minimize stress. After they left the site, efforts were made to collect samples from ten different individuals without repeating collections from the same animal. In total, 240 faecal samples were gathered between January 2023 and December 2023. The health, sex, and age of the sampled animals could not be determined from the observation distance. The samples were sealed in zip-lock bags, transported to the laboratory, where 5 grams of each sample were weighed, resealed, and stored at -20°C for later molecular analyses. 2.3 Molecular analysis Total DNA was extracted from faecal samples using the Quick-DNA Fecal/Soil Microbe MiniPrep Kit (Zymo Research, Irvine, CA, USA), following a protocol adapted from [ 25 ]. Frozen samples were thawed at room temperature and homogenized manually. From each sample, 1 g of feces was suspended in 800 µL BashingBead Buffer and 3,200 µL PBS, then mixed thoroughly. A 1,200 µL aliquot was transferred to a ZR BashingBead Lysis Tube and lysed using a Retsch MM200 mixer mill (1,800 rpm, 10 min). Each extraction batch (~ 30 samples) included a negative isolation control. DNA purification was completed according to the manufacturer’s instructions, and final eluates (50 µL) were stored at -20°C until further analysis. Specific primers and TaqMan probes targeting the ITS1 region of A. sidemi and the ITS2 region of Haemonchus spp. were used in a triplex real-time PCR assay, which also included a synthetic internal amplification control (IAC) to detect potential PCR inhibition. The reaction composition, primer/probe concentrations, and thermal cycling parameters were adopted from previously published protocols [ 25 ]. Briefly, each 20 µL reaction contained Luna Universal Probe qPCR Master Mix, 250 nM of each primer, 100 nM of FAM probe, 100 nM Cy5 probe, 200 nM of HEX probe, 0.4 U of Antarctic Thermolabile UDG (New England Biolabs, Ipswich, MA, United States), 1× 10 5 copies of IAC plasmid, 5 µL of template DNA, and PCR-grade water. Amplification was performed in duplicate on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) under following cycling conditions: 37°C for 10 min (carryover prevention), 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 57°C for 45 s, with final cooling at 40°C for 30 s. Data were analyzed using CFX Manager 3.0 software (Bio-Rad Laboratories). To enable detection and semi-quantification of both parasites, the relative abundance of DNA (expressed as plasmid copy number equivalents) was determined for each target using Ct values adjusted for inter-target differences with an empirically derived correction factor [ 26 ]. These values were subsequently interpolated against a standard curve generated from tenfold serial dilutions (ranging from 5 × 10⁷ to 5 × 10³ copies) of a plasmid construct containing the ITS2 sequence of H. contortus . 2.4 Data analysis For each sample, the presence or absence of A. sidemi and H. contortus (coded as 1 and 0) and the relative amounts of target DNA in positive samples were recorded and used as dependent variables. All data analyses were performed in R 4.3.2. Due to a higher-than-expected number of zero values in the dataset (zero inflation), the analysis was conducted in two steps. First, the probability of presence or absence was analyzed using a generalized linear mixed model (GLMM) with a binomial distribution and a logit link function. Second, to account for overdispersion, a GLMM with a negative binomial distribution was used to examine the variability in the relative abundance of parasite DNA in positive samples. Locality (site), season, and their interaction were included as fixed effects to assess whether the effect of site on parasite presence varied by season. Additionally, a random effect (1 | month:locality) was incorporated to account for month-to-month variability within each site. The models were implemented using the glmmTMB package, and data visualisation was performed in the ggplot2 package. The explanatory variable season was defined to categorize the study period into biologically meaningful timeframes. Winter (January–March) was characterized at Site A by continuous snow coverage and intensive additional feeding in shared feeders with deer, while at Site B, additional feeding was limited and restricted to the chamois-only feeder, with continuous grazing throughout. Spring (April–June) marked the transition to grazing at Site A and, at both sites, coincided with the peak phase of female gestation. The summer period (July–September) was defined by the presence of young individuals grazing and undergoing weaning at its peak, with mixed herds of chamois and red deer observed at Site B. Autumn (October–December) was characterized by the rutting season for males at both sites. Model diagnostics were performed using the DHARMa package. For samples that tested positive for both parasites simultaneously, a Spearman’s rank correlation test was conducted to assess potential interactions between the two species. Additionally, a Mann-Whitney U test was used to compare the relative abundance of A. sidemi DNA between samples with A. sidemi single infections and those with co-infections with H. contortus. 3. Results The presence of DNA from both parasite species ( A. sidemi and H. contortus ), was detected in the faeces of chamois at both monitored sites. H. contortus DNA was identified in 116 out of 240 samples, with a prevalence of 43.3% (95% CI = 36–49), while A. sidemi was found in only 12 samples (5%; 95% CI = 2–8). At Site A (Studený vrch), 62 out of 120 samples (51.7%; 95% CI = 42–60) tested positive for H. contortus , and 10 out of 120 (8.33%; 95% CI = 4–14) for A. sidemi . At the Česká Kamenice hunting ground (Site B), A. sidemi was detected in only two out of 120 samples (1.67%; 95% CI = 0.2–5.8), while H. contortus was found in 42 out of 120 samples (35%; 95% CI = 26–44). The monthly prevalence is presented in Fig. 1 . At Site A, A. sidemi was detected continuously from March to June, with the highest proportion of positive samples (4/10) recorded in March. The frequency of positive samples then gradually declined, with three cases confirmed in April, two in May, and one in June. No positive samples were recorded during the summer and autumn periods. At Site B, A. sidemi exhibited a similar seasonal pattern but at a lower frequency. During spring, it was detected only in April (1/10) and May (1/10), after which it was not observed. Due to the low prevalence of A. sidemi , the statistical model described in the Data Analysis section could not be applied to this species. As a result, neither the probability of presence nor the relative DNA quantity in positive samples was analyzed. However, at Site A—and to a limited extent at Site B—a rising trend in the relative amount of parasite DNA was observed in spring samples. H. contortus DNA was detected in samples from all months at both sites, except for March at Site B, with frequencies ranging from 10–90% (Fig. 1 ). The highest values were recorded at both sites in August. As shown in Table 1 , at Site B, the probability of H. contortus occurrence was significantly lower in the spring season (p < 0.05). Additionally, results indicated a lower prevalence during winter months at both sites, although this effect was not statistically significant (p = 0.069). Table 1 Results of the generalized linear mixed model (GLMM) for probability of Haemonchus contortus DNA presence. Response variable Factor Estimate ± SD Z-value p-value Presence/absence of H. contortus (Intercept) 0.134 ± 0.366 0.365 0.715 Winter -0.980 ± 0.541 -1.813 0.069 Spring*Locality B -2.005 ± 0.830 -2.414 0.016 Random effect Variance SD Month:locality 2.264e-09 4.759e-05 The relative abundance of H. contortus DNA varied seasonally, as shown in Fig. 2 . The highest values were detected in summer (p < 0.001), followed by a decline in autumn. In contrast, the lowest DNA levels were recorded in winter (p = 0.005) and spring (p < 0.001). The model results, summarized in Table 2 , revealed an interaction between spring and Site B, with significantly higher levels of H. contortus DNA in positive samples from Site B during the spring season (p < 0.001). However, this result was likely influenced by an extreme value and the low prevalence at this site during that period. Overall, no significant difference was found in the relative abundance of H. contortus DNA between the two sites (p = 0.231). Full model outputs and results of DHARMa residual diagnostics are available in Additional File 1. In total, eight samples tested positive for the DNA of both parasites simultaneously, while four samples were positive only for A. sidemi . A significant positive correlation was found between the relative abundance of A. sidemi and H. contortus DNA in co-infected samples (Spearman's rank correlation: ρ = 0.976, p = 0.0004). However, the comparison of A. sidemi DNA levels between single infections and co-infections showed no statistically significant difference (Mann-Whitney U test: W = 7, p = 0.153). Table 2 Results of the generalized linear mixed model (GLMM) for relative amount of Haemonchus contortus DNA in positive samples. Response variable Factor Estimate ± SD Z-value p-value Level of H. contortus DNA (Intercept) 12.42 ± 0.680 18.256 < 0.001 Spring -3.408 ± 0.905 -3.766 < 0.001 Summer 3.931 ± 0.868 4.528 < 0.001 Winter -2.617 ± 0.947 -2.762 0.006 Locality B -0.994 ± 0.831 -1.195 0.231 Spring*Locality B 8.083 ± 1.417 5.705 < 0.001 Random effect Variance SD Month:Locality 0.257 0.507 4. Discussion In this study, we investigated seasonal changes in contamination by eggs of two haematophagous nematodes, Haemonchus contortus and an invasive species A. sidemi , in the faeces of chamois at two sites in the Northern part of the Czech Republic. 4.1. Ashworthius sidemi The occurrence of A. sidemi in the studied chamois feacal samples was rather episodic as the DNA of this parasite was detected at Site A continuously from late winter through spring 2023, while at Site B, it was found only in April and May. To our knowledge, this is the first confirmed occurrence of A. sidemi in chamois in the CR. The only relevant previous record of A. sidemi infection in chamois comes from the Low Tatras (Slovakia), where it was detected in a single crossbred Tatra ( R. r. tatrica ) and Northern chamois ( R. r. rupicapra ) based on a digestive tract necropsy [ 13 ], while high prevalences were recorded in sympatric cervids. A. sidemi is generally considered a species typically associated with hosts from the subfamily Cervinae [ 15 ]. Although it has been confirmed as a generalist capable of infecting both free-living and domestic bovids [ 28 ], its occurrence in free-living bovids appears to be limited. The main exception appears to be the European bison, in which the parasite is well established, as evidenced by records from Poland and the CR [ 29 – 31 ]. The origin of chamois in the northern part of the CR is complex, as the founding individuals were imported from various zoos and private breeding facilities in the early 20th century. Since these animals were unlikely to have been in contact with sika deer, the primary host of A. sidemi , and this parasite has not been recorded in chamois in their countries of origin, such as Austria [ 32 ], or only appeared later due to its spread, as in Bavaria [ 14 ], it is unlikely that A. sidemi was introduced with the original chamois population. Additionally, molecular analysis recently confirmed the presence of A. sidemi eggs in red deer faeces at a site adjacent to the North Bohemian chamois population monitored in this study [ 20 ]. Given these factors, we hypothesize that the presence of A. sidemi in chamois from our study sites results from recent transmission from sympatric red deer, among which this parasite is actively spreading in the CR. Deer populations likely play a key role in facilitating their expansion into new areas in Europe [ 14 , 16 ]. This hypothesis is further supported by our finding of several nematodes, Spiculopteragia spiculoptera and Ostertagia leptospicularis , species typical of cervids [ 15 , 32 ] in the abomasum of a single chamois culled at the site A during a free hunting in autumn 2022 (unpublished data). When we compare both monitored sites, the slightly higher percentage of positivity and earlier onset of A. sidemi appear at Site A. This may be attributed to closer contact with red deer, as supplementary feeding was provided at shared feeders with red deer until April. In contrast, at Site B, chamois primarily relied on grazing, and only a feeding station was designed to restrict deer access, with supplementary feeding being less intensive. Additionally, increased stress levels could have led to higher susceptibility to the parasite. For instance, in Apennine chamois ( Rupicapra pyrenaica ornata ), faecal cortisol metabolite levels were found to rise during periods of habitat overlap with red deer [ 33 ]. Overall, the occurrence of A. sidemi in chamois was limited to the spring and winter seasons, contrasting with a previous longitudinal study on farmed fallow deer ( Dama dama ), where A. sidemi DNA was detected in pastures nearly year-round, except in the months following anthelmintic treatments [ 34 ]. However, the absence of A. sidemi DNA for most of the year does not necessarily indicate the parasite's absence in the host’s abomasum. Egg shedding may not directly reflect parasite load, as it can be influenced by reduced production or the presence of sexually immature stages [ 24 ]. A. sidemi is known to persist within the host for extended periods in its juvenile or larval stages [ 35 , 36 ]. Consistent with this, our dissection of two chamois culled in autumn 2023 revealed dozens of juvenile A. sidemi stages (unpublished data), suggesting its ongoing persistence despite the lack of detectable DNA in environmental samples. 4.2 Haemonchus contortus Haemonchus contortus was present in the samples of chamois from almost all monitored months. This parasite is globally recognized as a significant pathogen affecting small domestic ruminants, particularly sheep and goats. To a lesser extent, it has also been recorded in cattle. The presence of this parasite in wild ruminants, such as chamois, is typically associated with shared grazing areas in proximity to domestic ruminants [ 12 , 13 ] The low host specificity of H. contortus , combined with its ability to adapt to various ecological conditions, facilitates its circulation between domestic animals and mountain ungulates [ 10 , 37 ]. At the time of our study, shared grazing with domestic ruminants was not recorded; however, as shown by a recent study of H. contortus transmission between domestic ruminants and roe deer ( Capreolus capreolus ) in France the presence of this in wild ruminants is probably more closely related to long-term ecological co-occurrence rather than merely the actual intensity of contact with livestock [ 38 ]. The seasonal dynamics of Haemonchus contortus in ruminants are influenced by various environmental factors, particularly temperature and moisture, which significantly affect the life cycle and transmission [ 39 , 40 ]. To survive cold and dry periods, H. contortus larvae may enter a state of arrested development (hypobiosis), remaining in the abomasal tissue after being ingested by the host [ 41 , 42 ]. They can then reactivate and continue their development when environmental conditions become favorable. During this period, detecting eggs in faeces is typically challenging. Although the relative quantity of H. contortus DNA detected in our study during winter was significantly lower than in the reference (autumn) season, the number of positive samples indicated ongoing presence of reproducing stages. However, the likelihood of transmission during winter is limited as the low temperatures typical for this period rapidly reduce the ability to hatch and subsequent larval survival and dispersion [ 43 , 44 ]. The meteorological station in Česká Kamenice, located between the two study sites, recorded nearly 40 days with minimum temperatures below 0°C between January and March 2023. While at Site A, a continuous snow cover was recorded during this time, at Site B, the developmental stages were exposed to frost most of the time. Snow coverage can act as an insulating layer that can moderate soil temperatures and influence its moisture levels [ 45 ], which can explain the significant decline in positive samples during spring and the absence of the parasite at Site B in March. The highest prevalence and relative abundance of the parasite was found in the summer period, with a peak in August at both sites monitored. This was followed by a decline in autumn; however, the probability of parasite occurrence in autumn remained comparable to spring, while relative abundance was significantly higher than in spring samples. A similar pattern was observed in the United Kingdom, where historical data analysis showed that the number of ovine haemonchosis diagnoses peaked in late summer and persisted into early autumn [ 46 ]. Similarly, Citterio et al. [ 12 ] observed the dominance of H. contortus during the autumn hunting season in a five-year study of abomasal nematode communities in chamois in Italy. In our study, the parasite was detected in samples until January 2024. This timing of peak egg production in late summer and early autumn may enable the parasite to infect susceptible weaned young. If conditions remain favorable for hatching and larval development, these hosts may become infected and start shedding eggs within a few weeks. This could explain the slight increase in the relative amount of H. contortus DNA observed in our study in November. However, other host-related factors must also be considered. For instance, studies conducted in the western Italian Alps on chamois [ 47 , 48 ] reported male-biased lungworm larval counts in autumn, coinciding with the rutting season, with territorial males exhibiting higher lungworm burdens. Due to the way our data were obtained, it was not possible in this study to assess host factors affecting egg production, such as age groups and sex of the sample studied. Studies based on historical data and predictive models have shown that parasite transmission periods in temperate zones tend to increase with climate change [ 40 , 46 , 49 ]. Our study supports this trend, as we observed a relatively long period of potential infection. Minimum daily temperatures measured near the study sites remained above 0°C for most of November. However, to accurately assess the infectivity of the parasite, it would be necessary to monitor the microclimate of the vegetation at the grazing sites. 4.3. Possible interaction A comparison of seasonal dynamics of A. sidemi and H. contortus raises the question of potential interactions between these species. A meta-analysis of gastrointestinal nematode co-infections in sheep suggests that H. contortus generally exerts an antagonistic effect on co-infecting species [ 50 ]. However, DNA analysis of spring samples from co-infected chamois showed no direct negative interaction. Instead, A. sidemi and H. contortus exhibited a positive correlation in abundance, with no significant differences between A. sidemi -only infections and co-infections with H. contortus . Interestingly, the timing of A. sidemi disappearance from the samples coincided with a summer rapid increase in H. contortus levels at both sites. This was particularly surprising at site B, where the formation of mixed herds of chamois and red deer, a potential source of A. sidemi transmission, was recorded during this period. H. contortus is well known for its high biotic potential, allowing it to respond rapidly to even slight changes in external conditions [ 51 ]. In contrast, A. sidemi exhibited only a slight increase in pasture contamination during the summer, as reported in a previous study on its seasonal dynamics in fallow deer [ 34 ]. In the present study, the observed abundance of H. contortus DNA increased sharply during summer, likely due to rising temperatures that enhanced larval survival and development to infective stages. Subsequent large-scale infection by H. contortus may have triggered an immune response in the host, potentially reducing larvae infectivity and egg production of the A. sidemi . Such negative interactions of H. contortus on gastrointestinal nematodes have been previously observed, for example, on T. circumcincta [ 52 ] or Nematodirus battus [ 53 ]. However, these studies were conducted in laboratory settings using high doses of infective larvae, whereas our field study design allowed only relative quantification of potential infection. A more detailed assessment of the interaction between these two parasites will require further experimental research. Conclusions This study provides insights into seasonal dynamics of egg output for two important gastrointestinal nematodes, H. contortus and A. sidemi , in chamois in the northern CR. The findings show distinct seasonal patterns for both parasites, with H. contortus showing a clear seasonal variation, peaking during the summer months. The presence of this parasite throughout the year indicates ongoing infection cycles and suggests the potential for a prolonged period of transmission in a temperate environment. In contrast, A. sidemi displayed a more episodic occurrence, with DNA primarily detected during the winter and spring months. This limited presence may reflect the lower susceptibility of the atypical host or the parasite’s persistence in non-mating forms, such as juvenile or larval stages, which do not contribute to egg shedding. Our study represents the first confirmed record of A. sidemi in chamois in the CR, supporting the hypothesis that the parasite has likely been transmitted from sympatric red deer. Despite the absence of clear evidence of antagonistic interaction between H. contortus and A. sidemi , the observed patterns imply that shifts in parasite loads, such as the summer increase of H. contortus , may influence the dynamics of co-infection. This study contributes data on the ecology of gastrointestinal nematodes in wild ruminants, which can aid in the development of integrated management strategies to mitigate the impact of parasitic infections on both wild and domestic ruminant populations. Declarations Ethics approval and consent to participate Ethical approval for the studies involving animals was waived by the Institutional Ethics and Animal Welfare Committee of the Czech University of Life Sciences Prague. No additional ethical approvals were required for the collection of samples. The research was conducted using non-invasive methods, based solely on the examination of faecal samples, without direct contact with the animals or causing any increased disturbance. As the samples were collected in publicly accessible areas and without interfering with the animals, no special permits or licenses were necessary. All fieldwork was carried out in accordance with local legislation and institutional guidelines. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This research was financially supported by the Technology Agency of the Czech Republic, project No. SS05010070. Author Contribution J.M. analyzed the data, prepared visualizations, wrote, and drafted the manuscript. V.K. collected the samples, provided information about the study sites, and contributed to writing the manuscript. J.I. and L.Š. conducted the molecular analyses and contributed to writing the manuscript. J.V. designed the study, supervised the experiments, and provided advice on data interpretation. All authors read and approved the final version of the manuscript. Acknowledgement The authors would like to express their sincere gratitude to the owners and managers of the monitored hunting grounds for their cooperation and support. We also wish to thank the reviewers for their helpful comments and suggestions, which significantly improved the manuscript. Data Availability The dataset used and analysed during the current study are available from the corresponding author on reasonable request. References Corlatti L, Iacolina L, Safner T, Apollonio M, Buzan E, Ferretti F, et al. Past, present and future of chamois science. Wildl Biol. 2022. 10.1002/wlb3.01025 . Briedermann L, Still V. Die Gemse des Elbsandsteingebietes. Rupicapra r. rupicapra. Die Neue Brehm-Bücherei. Wittenberg Lutherstadt: A. Ziemsen; 1976. Martínková N, Zemanová B, Kranz A, Giménez MD, Hájková P. 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Albery GF, Kenyon F, Morris A, Morris S, Nussey DH, Pemberton JM. Seasonality of helminth infection in wild red deer varies between individuals and between parasite taxa. Parasitology. 2018;145:1410–20. Chambers A, Candy P, Green P, Sauermann C, Leathwick D. Seasonal output of gastrointestinal nematode eggs and lungworm larvae in farmed wapiti and red deer of New Zealand. Vet Parasitol. 2022;303:109660. Reslová N, Škorpiková L, Kyriánová IA, Vadlejch J, Höglund J, Skuce P, et al. The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays. Parasit Vectors. 2021;14:391. Beck HE, Zimmermann NE, McVicar TR, Vergopolan N, Berg A, Wood EF. Present and future Köppen-Geiger climate classification maps at 1-km resolution. Sci Data. 2018;5:180214. Ahrens CD. Meteorology today: an introduction to weather, climate, and the environment. Belmont: Cengage Learning Canada Inc; 2015. Kotrlá B, Kotrlý A, Koždoň O. Studies on the specifity of the nematode Ashworthius sidemi Schulz, 1933. Acta Vet Brno. 1976;45:123–26. Karbowiak G, Demiaszkiewicz AW, Pyziel AM, Wita I, Moskwa B, Werszko J, et al. The parasitic fauna of the European bison ( Bison bonasus ) (Linnaeus, 1758) and their impact on the conservation. Part 2 The structure and changes over time. Acta Parasitol. 2014;59:372–79. Kołodziej-Sobocińska M, Demiaszkiewicz AW, Pyziel AM, Kowalczyk R. Increased Parasitic Load in Captive-Released European Bison ( Bison bonasus ) has Important Implications for Reintroduction Programs. EcoHealth. 2018;15:467–71. Vadlejch J, Kyriánová IA, Rylková K, Zikmund M, Langrová I. Health risks associated with wild animal translocation: a case of the European bison and an alien parasite. Biol Invasions. 2017;19:1121–25. Wyrobisz-Papiewska A, Kowal J, Nosal P, Chovancová G, Rehbein S. Host specificity and species diversity of the Ostertagiinae Lopez-Neyra, 1947 in ruminants: A European perspective. Parasit Vectors. 2018;11:369. Formenti N, Viganó R, Fraquelli C, Trogu T, Bonfanti M, Lanfranchi P, et al. Increased hormonal stress response of Apennine chamois induced by interspecific interactions and anthropogenic disturbance. Eur J Wildl Res. 2018;64:68. Magdálek J, Škorpíková L, McFarland C, Vadlejch J. An alien parasite in a changing world – Ashworthius sidemi has lost its traditional seasonal dynamics. Front Vet Sci. 2023;10:1279073. Ovcharenko DA. Seasonal dynamics and development of Ashworthius sidemi (Trichostrongylidae), Oesophagostomum radiatum and O. venulosum (Strongylidae) of Cervus nippon hortulorum . Parazitologiya. 1968;2:470–4. Drózdz J, Demiaszkiewicz AW, Lachowicz J. Expansion of the Asiatic parasite Ashworthius sidemi (Nematoda, Trichostrongylidae) in wild ruminants in Polish territory. Parasitol Res. 2003;89:94–7. Beaumelle C, Toïgo C, Papet R, Benabed S, Beurier M, Bordes L, et al. Cross-transmission of resistant gastrointestinal nematodes between wildlife and transhumant sheep. Peer Community J. 2024;4:103. Beaumelle C, Redman E, Verheyden H, Jacquiet P, Bégoc N, Veyssière F, et al. Generalist nematodes dominate the nemabiome of roe deer in sympatry with sheep at a regional level. Int J Parasitol. 2022;52:751–61. Rinaldi L, Catelan D, Musella V, Cecconi L, Hertzberg H, Torgerson PR, et al. Haemonchus contortus : Spatial risk distribution for infection in sheep in Europe. Geospat Health. 2015;9:325–31. Rose H, Caminade C, Bolajoko MB, Phelan P, van Dijk J, Baylis M, et al. Climate-driven changes to the spatio-temporal distribution of the parasitic nematode, Haemonchus contortus , in sheep in Europe. Glob Chang Biol. 2016;22:1271–85. Gibbs HC. Hypobiosis in Parasitic Nematodes—An Update. Adv Parasitol. 1986;25:129–74. Sargison ND, Wilson DJ, Bartley DJ, Penny CD, Jackson F. Haemonchosis and teladorsagiosis in a Scottish sheep flock putatively associated with the overwintering of hypobiotic fourth stage larvae. Vet Parasitol. 2007;147:326–31. O’Connor LJ, Walkden-Brown SW, Kahn LP. Ecology of the free-living stages of major trichostrongylid parasites of sheep. Vet Parasitol. 2006;142:1–15. Rose H, Wang T, van Dijk J, Morgan ER. GLOWORM-FL: A simulation model of the effects of climate and climate change on the free-living stages of gastro-intestinal nematode parasites of ruminants. Ecol Modell. 2015;297:232–45. Wang T, Avramenko RW, Redman EM, Wit J, Gilleard JS, Colwell DD. High levels of third-stage larvae (L3) overwinter survival for multiple cattle gastrointestinal nematode species on western Canadian pastures as revealed by ITS2 rDNA metabarcoding. Parasit Vectors. 2020;13:458. van Dijk J, David GP, Baird G, Morgan ER. Back to the future: Developing hypotheses on the effects of climate change on ovine parasitic gastroenteritis from historical data. Vet Parasitol. 2008;158:73–84. Corlatti L, Béthaz S, von Hardenberg A, Bassano B, Palme R, Lovari S. Hormones, parasites and male mating tactics in Alpine chamois: Identifying the mechanisms of life history trade-offs. Anim Behav. 2012;84:1061–70. Corlatti L, Lorenzetti C, Bassano B. Parasitism and alternative reproductive tactics in Northern chamois. Ecol Evol. 2019;9:8749–58. McMahon C, Gordon AW, Edgar HWJ, Hanna REB, Brennan GP, Fairweather I. The effects of climate change on ovine parasitic gastroenteritis determined using veterinary surveillance and meteorological data for Northern Ireland over the period 1999–2009. Vet Parasitol. 2012;190:167–77. Evans MJ, Corripio-Miyar Y, Hayward A, Kenyon F, McNeilly TN, Nussey DH. Antagonism between co-infecting gastrointestinal nematodes: A meta-analysis of experimental infections in Sheep. Vet Parasitol. 2023;323:110053. van Dijk J, Sargison ND, Kenyon F, Skuce PJ. Climate change and infectious disease: helminthological challenges to farmed ruminants in temperate regions. Animal. 2010;4:377–92. Dobson RJ, Barnes EH, Birclijin SD, Gill JH. Researchnote the survival of Ostertagia circumcincta and Trichostrongylus colubriformis in faecal culture as a source of bias in apportioning egg counts to worm species. Int J Parasitol. 1992;22:1005–8. Mapes CJ, Coop RL. Effect of concureent and terminated infections of Haemonchus contortus on the development and reproductive capacity of Nematodirus battus . J Comp Pathol. 1971;81:479–92. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6529385","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451955007,"identity":"324b8c06-acfb-4102-943e-ce589c7a0bfd","order_by":0,"name":"Jan Magdálek","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3RsQrCMBSF4VMCugS6pii+gXClEJAWn0UotIug4OLo5lLoK0UCdimIW90qrn2ADgWtk5uxm2D++XzDTQCb7UcT2IBcOFUfQiBvz6gnIfUtmR7yS9lQ6Pu5BppWw83UZyKL1XaeUixlEcFJuYZQJqJWseCkQ6nWikFokLM3kHMdey09Qj+7g4E6wgy3yDI5jTgpSSLqyLIjAyOpWTCmyBflHcdUJVwUJnJObtd6t5hlWeRUTRtMjC8G8Pd3vLbctO8aVl+MbDab7a97AiH+QCmdR0dfAAAAAElFTkSuQmCC","orcid":"","institution":"Czech University of Life Sciences Prague","correspondingAuthor":true,"prefix":"","firstName":"Jan","middleName":"","lastName":"Magdálek","suffix":""},{"id":451955008,"identity":"47091ba2-d562-40c4-8f9c-39cc1615f1b7","order_by":1,"name":"Vojtěch Kasič","email":"","orcid":"","institution":"Czech University of Life Sciences Prague","correspondingAuthor":false,"prefix":"","firstName":"Vojtěch","middleName":"","lastName":"Kasič","suffix":""},{"id":451955009,"identity":"bd0c8dca-a4c0-4d0e-a3c6-5e63018a93a2","order_by":2,"name":"Jana Ilgová","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Ilgová","suffix":""},{"id":451955010,"identity":"73bad193-e0dc-4265-82b2-b5eacd30ded1","order_by":3,"name":"Lucie Škorpíková","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Lucie","middleName":"","lastName":"Škorpíková","suffix":""},{"id":451955011,"identity":"8f158d19-1e56-4f24-8155-90110fc0f8e8","order_by":4,"name":"Jaroslav Vadlejch","email":"","orcid":"","institution":"Czech University of Life Sciences Prague","correspondingAuthor":false,"prefix":"","firstName":"Jaroslav","middleName":"","lastName":"Vadlejch","suffix":""}],"badges":[],"createdAt":"2025-04-25 13:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6529385/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6529385/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-04992-6","type":"published","date":"2025-09-01T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82028681,"identity":"11bb8882-8712-43dc-bce0-baded69e3525","added_by":"auto","created_at":"2025-05-06 07:04:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80199,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of positive samples with 95% confidence intervals for \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e in the monitored months at locality A (Studený Vrch) and B (Česká Kamenice).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6529385/v1/74c302d091563da2ddc85960.png"},{"id":82028682,"identity":"4e0f46e3-2d8b-4247-b9c1-f2c7811d06e7","added_by":"auto","created_at":"2025-05-06 07:04:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":69620,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal variation in the amount of target DNA for \u003cem\u003eA. sidemi\u003c/em\u003e (red points) and \u003cem\u003eH. contortus\u003c/em\u003e (grey points) in positive samples collected at locality A (Studený Vrch) and B (Česká Kamenice) from January 2023 to January 2024. DNA levels are expressed as the plasmid standard copy number on a log-scaled y-axis.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6529385/v1/38d37557117fb42532efec92.png"},{"id":90827966,"identity":"37b00e88-361f-4e04-972f-cc0d0893cede","added_by":"auto","created_at":"2025-09-08 16:04:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":873729,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6529385/v1/df933621-7b63-4504-a9e0-81c734071a51.pdf"},{"id":82028690,"identity":"100c5d91-b84f-4055-bb69-daa4f8e4d856","added_by":"auto","created_at":"2025-05-06 07:04:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":702441,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6529385/v1/c84ed8514bdcb66bed964ca6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seasonal Dynamics and Potential Interactions of Haematophagous Abomasal Nematodes in two Chamois populations in the Czech Republic","fulltext":[{"header":"1. Background","content":"\u003cp\u003eThe chamois \u003cem\u003eRupicapra rupicapra\u003c/em\u003e is distributed to higher altitudes and specific, rocky terrain types; however, the species exhibits remarkable adaptability, allowing it to thrive in a wide range of mountainous environments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The chamois is considered a non-native species in the Czech Republic (CR) as the presence of two populations of chamois in the country has its origin in introductions that took place at the beginning of the 20th century to enrich the spectrum of the species for trophy hunting. These introductions were repeatedly carried from the alpine regions of Austria and Germany, as well as from zoos and game breeders in Austria and Switzerland [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Analysis of the mitochondrial control region shows that the current chamois populations in the CR, found in the northern part of the country (The Lusatian Mountains Protected Area (PLA) and Jesen\u0026iacute;ky Mountains, are most genetically related to northern chamois populations in the eastern Alps, specifically in the Ebensee and M\u0026uuml;rzsteg regions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As the mountain chamois is restricted to specific habitats and has low dispersal rates, gene flow is limited, resulting in genetically distinct populations. Introduced populations in Central Europe have shown lower genetic variability than their Alpine counterparts, and apparent consequences of founder effects and bottleneck events [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which could potentially increase their susceptibility to pathogens.\u003c/p\u003e \u003cp\u003eChamois host a variety of gastrointestinal parasites, with those infecting the abomasum being the most significant. While typically subclinical [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], these infections can negatively impact the host's fitness and welfare. A negative correlation between the species richness of abomasal nematodes and both skeletal development and nutritional status of alpine chamois in Italy has been observed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among these parasites, \u003cem\u003eHaemonchus contortus\u003c/em\u003e is considered the most important for chamois health [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although primarily associated with significant losses in small livestock farms worldwide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], this blood-sucking nematode has low host specificity and can spread between grazing livestock and wild ruminants, including northern chamois [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This parasite was identified as the direct cause of death in only 1.4% of postmortem-examined chamois in the Slovenian Alps [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, its broader impact should not be underestimated as subclinical infections may weaken individuals, making them more susceptible to other stressors. In the Italian Alps, \u003cem\u003eH. contortus\u003c/em\u003e was reported as a predominant abomasal parasite and suggested as a predisposing factor contributing to a pneumonia-related mass die-off [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, a close relative of the parasite \u003cem\u003eH. contortus\u003c/em\u003e from the subfamily Haemonchinae, \u003cem\u003eAshworthius sidemi\u003c/em\u003e, has recently been identified in a hybrid of alpine (\u003cem\u003eR. rupicapra rupicapra\u003c/em\u003e) and tatra chamois (\u003cem\u003eR. rupicapra tatrica\u003c/em\u003e) from the Low Tatras (Slovakia) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The original hosts of this nematode are thought to be the cervids of eastern Asia, but with the translocation of sika deer (\u003cem\u003eCervus nippon\u003c/em\u003e), it was inadvertently repeatedly introduced into central and eastern Europe and France, as reviewed by Rehbein et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It has successfully transferred from its original host to various local ruminant species and, in Europe, currently occurs locally with a high prevalence [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, with new hosts, there is likely to be further natural spread of the parasite into new territories [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The parasite was first recorded in the former Czechoslovakia in the 1970s at the L\u0026aacute;ny game reserve [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and recent molecular monitoring indicates that it is now most common in red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In that study, the \u003cem\u003eA. sidemi\u003c/em\u003e infection was also confirmed in red deer in the locality of chamois population occurrence in the northern part of the CR. Despite the different feeding strategies of red deer and chamois [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the diet composition observed in the other chamois locality, the Jesen\u0026iacute;ky Mountains, was almost identical for both species. Both diets were primarily composed of grasses, with smaller proportions of herbs and woody broad-leaved plants [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This overlap in ecological niches suggests the potential for transmission of the invasive \u003cem\u003eA. sidemi\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGastrointestinal nematode data from chamois have typically been collected through necropsy-based cross-sectional studies, e.g. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While these methods enable precise species identification and infection intensity estimates, they provide only a narrow insight into fluctuating infection status, as sampling is usually limited to the autumn hunting season.\u003c/p\u003e \u003cp\u003eAlternatively, coprological analysis offers a non-invasive way to monitor seasonal parasite output [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, this method does not allow species-level identification, as most strongylid parasite eggs are morphologically indistinguishable, limiting its ability to track the most pathogenic species. For these reasons, we adopted the real-time PCR method proposed by Reslov\u0026aacute; et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which enables reliable differentiation of the haematophagous nematodes \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e, as well as semi-quantification directly from faecal samples.\u003c/p\u003e \u003cp\u003eThe main goals of this study were to investigate the occurrence of the invasive species \u003cem\u003eA. sidemi\u003c/em\u003e and the important pathogen \u003cem\u003eH. contortus\u003c/em\u003e in a northern chamois population from the Lusatian Mountains PLA (CR) and to monitor seasonal variations in egg production of both parasites as well as explore potential interactions between them.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study site and animals\u003c/h2\u003e \u003cp\u003eThe chamois population in the northern part of the Czech Republic inhabits three protected areas: the Czech Switzerland National Park, the Lusatian Mountains PLA, and the Elbe Sandstones PLA. To monitor the occurrence and seasonal variation in \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e egg production, we selected two areas with the highest chamois presence: one in the Lusatian Mountains PLA and the other in the Elbe Sandstones PLA. These study sites were approximately seven km apart aerially, separated by fields, urban development, and a traffic road. Based on the K\u0026ouml;ppen\u0026ndash;Geiger climate classification [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the study area is characterized as a warm-summer humid continental climate, typical for Central Europe. This climate features clearly defined seasons, with warm but not excessively hot summers, long and cold winters, and generally lower levels of precipitation throughout the year [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The locality A, Studen\u0026yacute; Vrch, lies adjacent to the Studenec Hill (50\u0026deg;49\u0026prime;56\u0026Prime; N, 14\u0026deg;27\u0026prime;16\u0026Prime; E ) with its peak at an altitude of 737 m. The terrain consists of volcanic stony fields, partly refugial post-glacial basiphilous beech mixed fir-beech forests (alliance \u003cem\u003eFagion sylvaticae\u003c/em\u003e) and meadows dominated by false oatgrass (\u003cem\u003eArrhenatherum elatius\u003c/em\u003e) grazed by chamois during the vegetation period. Based on the census carried out by the local forestry service, the hunting ground covering this site was inhabited by 39 individuals in 2023.\u003c/p\u003e \u003cp\u003eFrom January to April, supplementary feeding of concentrates was carried out, while from March onwards, the movement to meadows and grazing was allowed. From the end of January until approximately mid-March, a continuous snow cover was observed at this site, except for one week at the end of February. Feeding took place on common feeders with red deer, whose numbers were estimated at 78 individuals on the hunting ground. During the grazing season, deer and chamois used the same meadows, but both species grazed mostly in separate parts. Mixed groups were not observed during the daytime.\u003c/p\u003e \u003cp\u003eThe second monitored site (locality B) was located in the Česk\u0026aacute; Kamenice hunting ground, specifically on Str\u0026aacute;žiště Hill (50\u0026deg;48\u0026prime;59\u0026Prime;N, 14\u0026deg;22\u0026prime;19\u0026Prime;E) at an altitude of 469 m. This area is characterised by generally lower elevation and is dominated by Man-made forests with high prevalence of \u003cem\u003ePicea\u003c/em\u003e sp. and vegetation from the \u003cem\u003eLuzulo-Fagetum\u003c/em\u003e association. The number of chamois at this site was estimated at 47 individuals by the local forestry service. Supplementary feeding was limited here to the winter season and occurred at feeding stations specially adjusted to be accessible only to chamois without disturbance by the red deer. Simultaneously, grazing was carried out all year round, with mixed groups of chamois and deer observed at this site. The total number of deer on this hunting ground was estimated at 126.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample collection\u003c/h2\u003e \u003cp\u003eFreshly excreted faecal samples were collected monthly from both sites. The animals were first observed with binoculars outside their flight distance to minimize stress. After they left the site, efforts were made to collect samples from ten different individuals without repeating collections from the same animal. In total, 240 faecal samples were gathered between January 2023 and December 2023. The health, sex, and age of the sampled animals could not be determined from the observation distance. The samples were sealed in zip-lock bags, transported to the laboratory, where 5 grams of each sample were weighed, resealed, and stored at -20\u0026deg;C for later molecular analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Molecular analysis\u003c/h2\u003e \u003cp\u003eTotal DNA was extracted from faecal samples using the Quick-DNA Fecal/Soil Microbe MiniPrep Kit (Zymo Research, Irvine, CA, USA), following a protocol adapted from [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Frozen samples were thawed at room temperature and homogenized manually. From each sample, 1 g of feces was suspended in 800 \u0026micro;L BashingBead Buffer and 3,200 \u0026micro;L PBS, then mixed thoroughly. A 1,200 \u0026micro;L aliquot was transferred to a ZR BashingBead Lysis Tube and lysed using a Retsch MM200 mixer mill (1,800 rpm, 10 min). Each extraction batch (~\u0026thinsp;30 samples) included a negative isolation control. DNA purification was completed according to the manufacturer\u0026rsquo;s instructions, and final eluates (50 \u0026micro;L) were stored at -20\u0026deg;C until further analysis.\u003c/p\u003e \u003cp\u003eSpecific primers and TaqMan probes targeting the \u003cem\u003eITS1\u003c/em\u003e region of \u003cem\u003eA. sidemi\u003c/em\u003e and the \u003cem\u003eITS2\u003c/em\u003e region of \u003cem\u003eHaemonchus\u003c/em\u003e spp. were used in a triplex real-time PCR assay, which also included a synthetic internal amplification control (IAC) to detect potential PCR inhibition. The reaction composition, primer/probe concentrations, and thermal cycling parameters were adopted from previously published protocols [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, each 20 \u0026micro;L reaction contained Luna Universal Probe qPCR Master Mix, 250 nM of each primer, 100 nM of FAM probe, 100 nM Cy5 probe, 200 nM of HEX probe, 0.4 U of Antarctic Thermolabile UDG (New England Biolabs, Ipswich, MA, United States), 1\u0026times; 10\u003csup\u003e5\u003c/sup\u003e copies of IAC plasmid, 5 \u0026micro;L of template DNA, and PCR-grade water. Amplification was performed in duplicate on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) under following cycling conditions: 37\u0026deg;C for 10 min (carryover prevention), 95\u0026deg;C for 2 min, followed by 40 cycles of 95\u0026deg;C for 15 s and 57\u0026deg;C for 45 s, with final cooling at 40\u0026deg;C for 30 s. Data were analyzed using CFX Manager 3.0 software (Bio-Rad Laboratories).\u003c/p\u003e \u003cp\u003eTo enable detection and semi-quantification of both parasites, the relative abundance of DNA (expressed as plasmid copy number equivalents) was determined for each target using Ct values adjusted for inter-target differences with an empirically derived correction factor [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. These values were subsequently interpolated against a standard curve generated from tenfold serial dilutions (ranging from 5 \u0026times; 10⁷ to 5 \u0026times; 10\u0026sup3; copies) of a plasmid construct containing the \u003cem\u003eITS2\u003c/em\u003e sequence of \u003cem\u003eH. contortus\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data analysis\u003c/h2\u003e \u003cp\u003eFor each sample, the presence or absence of \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e (coded as 1 and 0) and the relative amounts of target DNA in positive samples were recorded and used as dependent variables. All data analyses were performed in R 4.3.2. Due to a higher-than-expected number of zero values in the dataset (zero inflation), the analysis was conducted in two steps. First, the probability of presence or absence was analyzed using a generalized linear mixed model (GLMM) with a binomial distribution and a logit link function. Second, to account for overdispersion, a GLMM with a negative binomial distribution was used to examine the variability in the relative abundance of parasite DNA in positive samples. Locality (site), season, and their interaction were included as fixed effects to assess whether the effect of site on parasite presence varied by season. Additionally, a random effect (1 | month:locality) was incorporated to account for month-to-month variability within each site.\u003c/p\u003e \u003cp\u003eThe models were implemented using the \u003cem\u003eglmmTMB\u003c/em\u003e package, and data visualisation was performed in the ggplot2 package. The explanatory variable \u003cem\u003eseason\u003c/em\u003e was defined to categorize the study period into biologically meaningful timeframes. Winter (January\u0026ndash;March) was characterized at Site A by continuous snow coverage and intensive additional feeding in shared feeders with deer, while at Site B, additional feeding was limited and restricted to the chamois-only feeder, with continuous grazing throughout. Spring (April\u0026ndash;June) marked the transition to grazing at Site A and, at both sites, coincided with the peak phase of female gestation. The summer period (July\u0026ndash;September) was defined by the presence of young individuals grazing and undergoing weaning at its peak, with mixed herds of chamois and red deer observed at Site B. Autumn (October\u0026ndash;December) was characterized by the rutting season for males at both sites. Model diagnostics were performed using the DHARMa package. For samples that tested positive for both parasites simultaneously, a Spearman\u0026rsquo;s rank correlation test was conducted to assess potential interactions between the two species. Additionally, a Mann-Whitney U test was used to compare the relative abundance of \u003cem\u003eA. sidemi\u003c/em\u003e DNA between samples with \u003cem\u003eA. sidemi\u003c/em\u003e single infections and those with co-infections with \u003cem\u003eH. contortus.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe presence of DNA from both parasite species (\u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e), was detected in the faeces of chamois at both monitored sites. \u003cem\u003eH. contortus\u003c/em\u003e DNA was identified in 116 out of 240 samples, with a prevalence of 43.3% (95% CI\u0026thinsp;=\u0026thinsp;36\u0026ndash;49), while \u003cem\u003eA. sidemi\u003c/em\u003e was found in only 12 samples (5%; 95% CI\u0026thinsp;=\u0026thinsp;2\u0026ndash;8). At Site A (Studen\u0026yacute; vrch), 62 out of 120 samples (51.7%; 95% CI\u0026thinsp;=\u0026thinsp;42\u0026ndash;60) tested positive for \u003cem\u003eH. contortus\u003c/em\u003e, and 10 out of 120 (8.33%; 95% CI\u0026thinsp;=\u0026thinsp;4\u0026ndash;14) for \u003cem\u003eA. sidemi\u003c/em\u003e. At the Česk\u0026aacute; Kamenice hunting ground (Site B), \u003cem\u003eA. sidemi\u003c/em\u003e was detected in only two out of 120 samples (1.67%; 95% CI\u0026thinsp;=\u0026thinsp;0.2\u0026ndash;5.8), while \u003cem\u003eH. contortus\u003c/em\u003e was found in 42 out of 120 samples (35%; 95% CI\u0026thinsp;=\u0026thinsp;26\u0026ndash;44). The monthly prevalence is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt Site A, \u003cem\u003eA. sidemi\u003c/em\u003e was detected continuously from March to June, with the highest proportion of positive samples (4/10) recorded in March. The frequency of positive samples then gradually declined, with three cases confirmed in April, two in May, and one in June. No positive samples were recorded during the summer and autumn periods. At Site B, \u003cem\u003eA. sidemi\u003c/em\u003e exhibited a similar seasonal pattern but at a lower frequency. During spring, it was detected only in April (1/10) and May (1/10), after which it was not observed. Due to the low prevalence of \u003cem\u003eA. sidemi\u003c/em\u003e, the statistical model described in the Data Analysis section could not be applied to this species. As a result, neither the probability of presence nor the relative DNA quantity in positive samples was analyzed. However, at Site A\u0026mdash;and to a limited extent at Site B\u0026mdash;a rising trend in the relative amount of parasite DNA was observed in spring samples.\u003c/p\u003e \u003cp\u003e \u003cem\u003eH. contortus\u003c/em\u003e DNA was detected in samples from all months at both sites, except for March at Site B, with frequencies ranging from 10\u0026ndash;90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The highest values were recorded at both sites in August. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, at Site B, the probability of \u003cem\u003eH. contortus\u003c/em\u003e occurrence was significantly lower in the spring season (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, results indicated a lower prevalence during winter months at both sites, although this effect was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.069).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of the generalized linear mixed model (GLMM) for probability of \u003cem\u003eHaemonchus contortus\u003c/em\u003e DNA presence.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse variable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstimate\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZ-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePresence/absence of \u003cem\u003eH. contortus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.715\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.980\u0026thinsp;\u0026plusmn;\u0026thinsp;0.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-1.813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring*Locality B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.005\u0026thinsp;\u0026plusmn;\u0026thinsp;0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-2.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRandom effect\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth:locality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.264e-09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.759e-05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe relative abundance of \u003cem\u003eH. contortus\u003c/em\u003e DNA varied seasonally, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The highest values were detected in summer (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), followed by a decline in autumn. In contrast, the lowest DNA levels were recorded in winter (p\u0026thinsp;=\u0026thinsp;0.005) and spring (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The model results, summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, revealed an interaction between spring and Site B, with significantly higher levels of \u003cem\u003eH. contortus\u003c/em\u003e DNA in positive samples from Site B during the spring season (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, this result was likely influenced by an extreme value and the low prevalence at this site during that period. Overall, no significant difference was found in the relative abundance of \u003cem\u003eH. contortus\u003c/em\u003e DNA between the two sites (p\u0026thinsp;=\u0026thinsp;0.231). Full model outputs and results of DHARMa residual diagnostics are available in Additional File 1. In total, eight samples tested positive for the DNA of both parasites simultaneously, while four samples were positive only for \u003cem\u003eA. sidemi\u003c/em\u003e. A significant positive correlation was found between the relative abundance of \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e DNA in co-infected samples (Spearman's rank correlation: ρ\u0026thinsp;=\u0026thinsp;0.976, p\u0026thinsp;=\u0026thinsp;0.0004). However, the comparison of \u003cem\u003eA. sidemi\u003c/em\u003e DNA levels between single infections and co-infections showed no statistically significant difference (Mann-Whitney U test: W\u0026thinsp;=\u0026thinsp;7, p\u0026thinsp;=\u0026thinsp;0.153).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of the generalized linear mixed model (GLMM) for relative amount of \u003cem\u003eHaemonchus contortus\u003c/em\u003e DNA in positive samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse variable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFactor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstimate\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZ-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eLevel of \u003cem\u003eH. contortus\u003c/em\u003e DNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Intercept)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.408\u0026thinsp;\u0026plusmn;\u0026thinsp;0.905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-3.766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.931\u0026thinsp;\u0026plusmn;\u0026thinsp;0.868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.528\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWinter\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.617\u0026thinsp;\u0026plusmn;\u0026thinsp;0.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-2.762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocality B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.994\u0026thinsp;\u0026plusmn;\u0026thinsp;0.831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-1.195\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpring*Locality B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.083\u0026thinsp;\u0026plusmn;\u0026thinsp;1.417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRandom effect\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonth:Locality\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.257\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we investigated seasonal changes in contamination by eggs of two haematophagous nematodes, \u003cem\u003eHaemonchus contortus\u003c/em\u003e and an invasive species \u003cem\u003eA. sidemi\u003c/em\u003e, in the faeces of chamois at two sites in the Northern part of the Czech Republic.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Ashworthius sidemi\u003c/h2\u003e \u003cp\u003eThe occurrence of \u003cem\u003eA. sidemi\u003c/em\u003e in the studied chamois feacal samples was rather episodic as the DNA of this parasite was detected at Site A continuously from late winter through spring 2023, while at Site B, it was found only in April and May. To our knowledge, this is the first confirmed occurrence of \u003cem\u003eA. sidemi\u003c/em\u003e in chamois in the CR. The only relevant previous record of \u003cem\u003eA. sidemi\u003c/em\u003e infection in chamois comes from the Low Tatras (Slovakia), where it was detected in a single crossbred Tatra (\u003cem\u003eR. r. tatrica\u003c/em\u003e) and Northern chamois (\u003cem\u003eR. r. rupicapra\u003c/em\u003e) based on a digestive tract necropsy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while high prevalences were recorded in sympatric cervids. \u003cem\u003eA. sidemi\u003c/em\u003e is generally considered a species typically associated with hosts from the subfamily Cervinae [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although it has been confirmed as a generalist capable of infecting both free-living and domestic bovids [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], its occurrence in free-living bovids appears to be limited. The main exception appears to be the European bison, in which the parasite is well established, as evidenced by records from Poland and the CR [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe origin of chamois in the northern part of the CR is complex, as the founding individuals were imported from various zoos and private breeding facilities in the early 20th century. Since these animals were unlikely to have been in contact with sika deer, the primary host of \u003cem\u003eA. sidemi\u003c/em\u003e, and this parasite has not been recorded in chamois in their countries of origin, such as Austria [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], or only appeared later due to its spread, as in Bavaria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it is unlikely that \u003cem\u003eA. sidemi\u003c/em\u003e was introduced with the original chamois population. Additionally, molecular analysis recently confirmed the presence of \u003cem\u003eA. sidemi\u003c/em\u003e eggs in red deer faeces at a site adjacent to the North Bohemian chamois population monitored in this study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these factors, we hypothesize that the presence of \u003cem\u003eA. sidemi\u003c/em\u003e in chamois from our study sites results from recent transmission from sympatric red deer, among which this parasite is actively spreading in the CR. Deer populations likely play a key role in facilitating their expansion into new areas in Europe [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This hypothesis is further supported by our finding of several nematodes, \u003cem\u003eSpiculopteragia spiculoptera\u003c/em\u003e and \u003cem\u003eOstertagia leptospicularis\u003c/em\u003e, species typical of cervids [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] in the abomasum of a single chamois culled at the site A during a free hunting in autumn 2022 (unpublished data).\u003c/p\u003e \u003cp\u003eWhen we compare both monitored sites, the slightly higher percentage of positivity and earlier onset of \u003cem\u003eA. sidemi\u003c/em\u003e appear at Site A. This may be attributed to closer contact with red deer, as supplementary feeding was provided at shared feeders with red deer until April. In contrast, at Site B, chamois primarily relied on grazing, and only a feeding station was designed to restrict deer access, with supplementary feeding being less intensive.\u003c/p\u003e \u003cp\u003eAdditionally, increased stress levels could have led to higher susceptibility to the parasite. For instance, in Apennine chamois (\u003cem\u003eRupicapra pyrenaica ornata\u003c/em\u003e), faecal cortisol metabolite levels were found to rise during periods of habitat overlap with red deer [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, the occurrence of \u003cem\u003eA. sidemi\u003c/em\u003e in chamois was limited to the spring and winter seasons, contrasting with a previous longitudinal study on farmed fallow deer (\u003cem\u003eDama dama\u003c/em\u003e), where \u003cem\u003eA. sidemi\u003c/em\u003e DNA was detected in pastures nearly year-round, except in the months following anthelmintic treatments [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the absence of \u003cem\u003eA. sidemi\u003c/em\u003e DNA for most of the year does not necessarily indicate the parasite's absence in the host’s abomasum. Egg shedding may not directly reflect parasite load, as it can be influenced by reduced production or the presence of sexually immature stages [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eA. sidemi\u003c/em\u003e is known to persist within the host for extended periods in its juvenile or larval stages [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Consistent with this, our dissection of two chamois culled in autumn 2023 revealed dozens of juvenile \u003cem\u003eA. sidemi\u003c/em\u003e stages (unpublished data), suggesting its ongoing persistence despite the lack of detectable DNA in environmental samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Haemonchus contortus\u003c/h2\u003e \u003cp\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e was present in the samples of chamois from almost all monitored months. This parasite is globally recognized as a significant pathogen affecting small domestic ruminants, particularly sheep and goats. To a lesser extent, it has also been recorded in cattle. The presence of this parasite in wild ruminants, such as chamois, is typically associated with shared grazing areas in proximity to domestic ruminants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] The low host specificity of \u003cem\u003eH. contortus\u003c/em\u003e, combined with its ability to adapt to various ecological conditions, facilitates its circulation between domestic animals and mountain ungulates [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. At the time of our study, shared grazing with domestic ruminants was not recorded; however, as shown by a recent study of \u003cem\u003eH. contortus\u003c/em\u003e transmission between domestic ruminants and roe deer (\u003cem\u003eCapreolus capreolus\u003c/em\u003e) in France the presence of this in wild ruminants is probably more closely related to long-term ecological co-occurrence rather than merely the actual intensity of contact with livestock [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe seasonal dynamics of \u003cem\u003eHaemonchus contortus\u003c/em\u003e in ruminants are influenced by various environmental factors, particularly temperature and moisture, which significantly affect the life cycle and transmission [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo survive cold and dry periods, \u003cem\u003eH. contortus\u003c/em\u003e larvae may enter a state of arrested development (hypobiosis), remaining in the abomasal tissue after being ingested by the host [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. They can then reactivate and continue their development when environmental conditions become favorable. During this period, detecting eggs in faeces is typically challenging. Although the relative quantity of \u003cem\u003eH. contortus\u003c/em\u003e DNA detected in our study during winter was significantly lower than in the reference (autumn) season, the number of positive samples indicated ongoing presence of reproducing stages. However, the likelihood of transmission during winter is limited as the low temperatures typical for this period rapidly reduce the ability to hatch and subsequent larval survival and dispersion [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The meteorological station in Česká Kamenice, located between the two study sites, recorded nearly 40 days with minimum temperatures below 0°C between January and March 2023. While at Site A, a continuous snow cover was recorded during this time, at Site B, the developmental stages were exposed to frost most of the time. Snow coverage can act as an insulating layer that can moderate soil temperatures and influence its moisture levels [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], which can explain the significant decline in positive samples during spring and the absence of the parasite at Site B in March.\u003c/p\u003e \u003cp\u003eThe highest prevalence and relative abundance of the parasite was found in the summer period, with a peak in August at both sites monitored. This was followed by a decline in autumn; however, the probability of parasite occurrence in autumn remained comparable to spring, while relative abundance was significantly higher than in spring samples. A similar pattern was observed in the United Kingdom, where historical data analysis showed that the number of ovine haemonchosis diagnoses peaked in late summer and persisted into early autumn [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Similarly, Citterio et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] observed the dominance of \u003cem\u003eH. contortus\u003c/em\u003e during the autumn hunting season in a five-year study of abomasal nematode communities in chamois in Italy. In our study, the parasite was detected in samples until January 2024. This timing of peak egg production in late summer and early autumn may enable the parasite to infect susceptible weaned young. If conditions remain favorable for hatching and larval development, these hosts may become infected and start shedding eggs within a few weeks. This could explain the slight increase in the relative amount of \u003cem\u003eH. contortus\u003c/em\u003e DNA observed in our study in November. However, other host-related factors must also be considered. For instance, studies conducted in the western Italian Alps on chamois [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] reported male-biased lungworm larval counts in autumn, coinciding with the rutting season, with territorial males exhibiting higher lungworm burdens. Due to the way our data were obtained, it was not possible in this study to assess host factors affecting egg production, such as age groups and sex of the sample studied.\u003c/p\u003e \u003cp\u003eStudies based on historical data and predictive models have shown that parasite transmission periods in temperate zones tend to increase with climate change [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Our study supports this trend, as we observed a relatively long period of potential infection. Minimum daily temperatures measured near the study sites remained above 0°C for most of November. However, to accurately assess the infectivity of the parasite, it would be necessary to monitor the microclimate of the vegetation at the grazing sites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Possible interaction\u003c/h2\u003e \u003cp\u003eA comparison of seasonal dynamics of \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e raises the question of potential interactions between these species. A meta-analysis of gastrointestinal nematode co-infections in sheep suggests that \u003cem\u003eH. contortus\u003c/em\u003e generally exerts an antagonistic effect on co-infecting species [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, DNA analysis of spring samples from co-infected chamois showed no direct negative interaction.\u003c/p\u003e \u003cp\u003eInstead, \u003cem\u003eA. sidemi\u003c/em\u003e and \u003cem\u003eH. contortus\u003c/em\u003e exhibited a positive correlation in abundance, with no significant differences between \u003cem\u003eA. sidemi\u003c/em\u003e-only infections and co-infections with \u003cem\u003eH. contortus\u003c/em\u003e. Interestingly, the timing of \u003cem\u003eA. sidemi\u003c/em\u003e disappearance from the samples coincided with a summer rapid increase in \u003cem\u003eH. contortus\u003c/em\u003e levels at both sites. This was particularly surprising at site B, where the formation of mixed herds of chamois and red deer, a potential source of \u003cem\u003eA. sidemi\u003c/em\u003e transmission, was recorded during this period. \u003cem\u003eH. contortus\u003c/em\u003e is well known for its high biotic potential, allowing it to respond rapidly to even slight changes in external conditions [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In contrast, \u003cem\u003eA. sidemi\u003c/em\u003e exhibited only a slight increase in pasture contamination during the summer, as reported in a previous study on its seasonal dynamics in fallow deer [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the present study, the observed abundance of \u003cem\u003eH. contortus\u003c/em\u003e DNA increased sharply during summer, likely due to rising temperatures that enhanced larval survival and development to infective stages. Subsequent large-scale infection by \u003cem\u003eH. contortus\u003c/em\u003e may have triggered an immune response in the host, potentially reducing larvae infectivity and egg production of the \u003cem\u003eA. sidemi\u003c/em\u003e. Such negative interactions of \u003cem\u003eH. contortus\u003c/em\u003e on gastrointestinal nematodes have been previously observed, for example, on \u003cem\u003eT. circumcincta\u003c/em\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] or \u003cem\u003eNematodirus battus\u003c/em\u003e [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. However, these studies were conducted in laboratory settings using high doses of infective larvae, whereas our field study design allowed only relative quantification of potential infection. A more detailed assessment of the interaction between these two parasites will require further experimental research.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides insights into seasonal dynamics of egg output for two important gastrointestinal nematodes, \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eA. sidemi\u003c/em\u003e, in chamois in the northern CR. The findings show distinct seasonal patterns for both parasites, with \u003cem\u003eH. contortus\u003c/em\u003e showing a clear seasonal variation, peaking during the summer months. The presence of this parasite throughout the year indicates ongoing infection cycles and suggests the potential for a prolonged period of transmission in a temperate environment. In contrast, \u003cem\u003eA. sidemi\u003c/em\u003e displayed a more episodic occurrence, with DNA primarily detected during the winter and spring months. This limited presence may reflect the lower susceptibility of the atypical host or the parasite’s persistence in non-mating forms, such as juvenile or larval stages, which do not contribute to egg shedding. Our study represents the first confirmed record of \u003cem\u003eA. sidemi\u003c/em\u003e in chamois in the CR, supporting the hypothesis that the parasite has likely been transmitted from sympatric red deer. Despite the absence of clear evidence of antagonistic interaction between \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eA. sidemi\u003c/em\u003e, the observed patterns imply that shifts in parasite loads, such as the summer increase of \u003cem\u003eH. contortus\u003c/em\u003e, may influence the dynamics of co-infection. This study contributes data on the ecology of gastrointestinal nematodes in wild ruminants, which can aid in the development of integrated management strategies to mitigate the impact of parasitic infections on both wild and domestic ruminant populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e Ethical approval for the studies involving animals was waived by the Institutional Ethics and Animal Welfare Committee of the Czech University of Life Sciences Prague. No additional ethical approvals were required for the collection of samples. The research was conducted using non-invasive methods, based solely on the examination of faecal samples, without direct contact with the animals or causing any increased disturbance. As the samples were collected in publicly accessible areas and without interfering with the animals, no special permits or licenses were necessary. All fieldwork was carried out in accordance with local legislation and institutional guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was financially supported by the Technology Agency of the Czech Republic, project No. SS05010070.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.M. analyzed the data, prepared visualizations, wrote, and drafted the manuscript. V.K. collected the samples, provided information about the study sites, and contributed to writing the manuscript. J.I. and L.Š. conducted the molecular analyses and contributed to writing the manuscript. J.V. designed the study, supervised the experiments, and provided advice on data interpretation. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to express their sincere gratitude to the owners and managers of the monitored hunting grounds for their cooperation and support. We also wish to thank the reviewers for their helpful comments and suggestions, which significantly improved the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe dataset used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCorlatti L, Iacolina L, Safner T, Apollonio M, Buzan E, Ferretti F, et al. Past, present and future of chamois science. 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J Comp Pathol. 1971;81:479\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chamois, Haemonchus contortus, Ashworthius sidemi, Real-time PCR, Prevalence, Epidemiology","lastPublishedDoi":"10.21203/rs.3.rs-6529385/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6529385/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground:\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePathogenic blood-feeding nematodes, such as \u003cem\u003eHaemonchus contortus\u003c/em\u003e and the invasive \u003cem\u003eAshworthius sidemi\u003c/em\u003e, infect a wide range of wild and domestic ruminants. While the spread of \u003cem\u003eA. sidemi\u003c/em\u003e among European cervids has been studied, its presence in chamois (\u003cem\u003eRupicapra rupicapra\u003c/em\u003e) remains poorly documented. Conversely, \u003cem\u003eH. contortus\u003c/em\u003e is known to infect chamois, but previous research has relied mainly on cross-sectional necropsy studies, offering only a limited view of infection dynamics. In this study, we used a longitudinal molecular approach to assess the seasonal occurrence and transmission patterns of \u003cem\u003eH. contortus\u003c/em\u003e and \u003cem\u003eA. sidemi\u003c/em\u003e in a chamois population from the northern Czech Republic. From January to December 2023, we collected faecal samples at monthly intervals from two localities. Multiplex real-time PCR was subsequently used for the detection and semi-quantification of DNA from both nematode species.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults:\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e DNA was detected in 43.3% of samples, with its presence recorded nearly year-round. Its prevalence and relative abundance peaked in summer and remained high throughout autumn. \u003cem\u003eAshworthius sidemi\u003c/em\u003e was identified in chamois in the Czech Republic for the first time, likely due to recent spillover from red deer (\u003cem\u003eCervus elaphus\u003c/em\u003e). However, it was found in only 5% of samples, with its occurrence restricted to late winter and spring. The seasonal disappearance of \u003cem\u003eA. sidemi\u003c/em\u003e coincided with the increase in \u003cem\u003eH. contortus\u003c/em\u003e abundance, suggesting a possible negative interaction between these species occupying the same ecological niche.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion:\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur findings indicate a prolonged transmission window for \u003cem\u003eH. contortus\u003c/em\u003e, which may expand further with climate change. In contrast, \u003cem\u003eA. sidemi\u003c/em\u003e appears to be an incidental parasite in chamois, and its long-term persistence in this atypical host without continued contact with cervids remains uncertain. These insights, which are rare for wild ruminants, contribute to a better understanding of parasite epidemiology and host-parasite interactions in free-living populations.\u003c/p\u003e","manuscriptTitle":"Seasonal Dynamics and Potential Interactions of Haematophagous Abomasal Nematodes in two Chamois populations in the Czech Republic","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 07:04:18","doi":"10.21203/rs.3.rs-6529385/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-23T10:07:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T08:54:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2025-07-17T12:11:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217008602069576537255340004121032169917","date":"2025-06-25T03:12:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-19T11:31:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291287008649889834875661180053749152430","date":"2025-05-12T13:25:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199029065925559909534302656918119992913","date":"2025-05-11T18:11:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-06T11:55:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-05T08:34:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308882406863088824147807142236059923174","date":"2025-05-05T07:37:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72378184267441099284926927046638180884","date":"2025-05-05T04:37:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199029065925559909534302656918119992913","date":"2025-05-01T09:23:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T18:23:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T07:47:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-29T13:57:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-29T09:52:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-04-29T09:51:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"82f8bcfe-b72d-4075-a2b8-6f642bf468d2","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:01:20+00:00","versionOfRecord":{"articleIdentity":"rs-6529385","link":"https://doi.org/10.1186/s12917-025-04992-6","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-09-01 15:57:57","publishedOnDateReadable":"September 1st, 2025"},"versionCreatedAt":"2025-05-06 07:04:18","video":"","vorDoi":"10.1186/s12917-025-04992-6","vorDoiUrl":"https://doi.org/10.1186/s12917-025-04992-6","workflowStages":[]},"version":"v1","identity":"rs-6529385","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6529385","identity":"rs-6529385","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}