First Molecular Detection and Phylogeny of Trichostrongylus axei and Spiculopteragia houdemeri From Indonesian Deer

Introduction
Gastrointestinal parasitic infections are a significant health concern affecting wild and domestic animals. Parasitic nematodes are common gut parasites in deer populations, contributing to clinical signs, such as weight loss, anemia, diarrhea, and, in severe cases, mortality (Modabbernia et al., 2021; Khattak et al., 2023; Yan et al., 2023). In addition to their direct effects on individual health, these parasites may also impact reproductive quality and population dynamics, especially in areas where deer are managed for conservation (Phetla et al., 2024). In Indonesia, studies on parasitic infections in deer remain limited, despite the country's rich biodiversity and the presence of both native and introduced deer species (Rahman et al., 2020).
Several species of gastrointestinal nematodes have been documented infecting deer. Common genera include Haemonchus, Ostertagia, Trichostrongylus, Cooperia, Oesophagostomum, and Spiculopteragia (Davidson et al., 2014; Lyons et al., 2024). These parasites are transmitted through the faecal-oral route influenced by environmental conditions, host density, and management practices (Morgan et al., 2013; Roeber et al., 2013a). In Indonesia, although some studies have documented Haemonchus contortus, Oesophagostomum spp., Trichostrongylus spp., and Fasciola spp. infections in domestic and wild ruminants (Puspitasari et al., 2016; Purnama et al., 2021; Arif et al., 2024), specific information on nematode infections in deer remains rare.
Spiculopteragia spp. are trichostrongyle nematodes that primarily inhabit the abomasum of cervids and other ruminants. Infections with Spiculopteragia houdemeri have been reported from several regions, including Austria, Japan, and France, mainly affecting sika deer (Cervus nippon centralis), red deer (Cervus elaphus), and roe deer (Capreolus capreolus) (Patrelle et al., 2014; Inoue et al., 2022). Similarly, Trichostrongylus axei is a cosmopolitan nematode that infects a broad range of hosts, such as cattle, sheep, goats, and various wildlife species, including deer (ten Doesschate et al., 2017; Halvarsson et al., 2022). Residing in the abomasum and sometimes the stomach, T. axei causes parasitic gastritis that may lead to decreased feed conversion efficiency and weight loss (Bhat et al., 2023). Although its presence is well-documented in parts of Europe, North America, and Asia (Davidson et al., 2014; Inoue et al., 2022), reports from Southeast Asia, particularly Indonesia, are still limited. 
Molecular techniques have greatly enhanced the accuracy of parasite identification, particularly through the use of ribosomal DNA regions, such as internal transcribed spacer 2 (ITS2) (Meshgi et al., 2015; Workentine et al., 2020; Rojas et al., 2025). Phylogenetic analysis based on these markers can provide valuable insights into parasite diversity, evolutionary relationships, and biogeographical patterns (Kumar et al., 2021).
This study aimed to provide the first molecular identification and phylogenetic characterization of T. axei and S. houdemeri in Indonesian deer. Polymerase chain reaction (PCR) and ITS2 sequencing were used for species confirmation. Phylogenetic trees were constructed to examine the evolutionary relationships. This research is expected to improve understanding of parasitic nematode genetics and distribution in Southeast Asia and support future epidemiological studies.

​​​​​​​Materials and Methods
Study area and sample collection
This study was conducted by collecting deer fecal samples from 13 breeding sites across West Java, Indonesia (Figure 1).

Fecal samples were collected immediately after defecation to ensure sample freshness. The Timor deer (Cervus timorensis) is classified as vulnerable on the IUCN red list (IUCN, 2018) and is legally protected in Indonesia, while the spotted deer (Axis axis) is listed as least concern globally but also protected under national law (IUCN, 2025). Samples were transported in a cooling box to the Helminthology Laboratory, School of Veterinary Medicine and Biomedical Sciences, IPB University. The samples were kept in a refrigerator at 4 °C for further analysis. 

Fecal examination and larval culture
Fecal samples were examined qualitatively using a flotation technique, which separates parasite eggs from fecal debris based on differences in specific gravity. This method allows clear visualization of eggs under a microscope for morphological identification. A quantitative McMaster technique was used to estimate egg counts per gram of feces (EPG), providing an assessment of infection intensity (Zajac et al., 2021). In addition, larval cultures were established using the Baermann technique and harvested on the seventh day post-culture. The harvested larvae were washed with distilled water, then transferred into 200 μL microtubes with 10 μL of distilled water allocated per larva. The tubes were stored at 1-4 °C and kept refrigerated until DNA extraction.

DNA extraction and molecular analysis (PCR and sequencing)
DNA extraction was performed using a lysis buffer composed of a direct PCR kit, 1 M dithiothreitol (DTT), and proteinase K, in a ratio of 25:1:1 per sample. A total of 10 μL of the lysis solution was added to each larval sample and incubated at 60 °C for 60 minutes, followed by 95 °C for 10 minutes.
PCR amplification targeted the ITS2 region using generic forward and reverse primers as described by Bisset et al.: Forward primer ITS2GF (5’-CACGAATTGCAGACGCTTAG-3’) and reverse primer ITS2GR (5’-GCTAAATGATATGCTTAAGTTCAGC-3’), producing an expected product size of 370–398 bp. The PCR reaction was performed in a final volume of 30 μL, consisting of 15 μL GoTaq® Green Master Mix (Promega, Madison, WI, USA), 1.5 μL of 10 μM of each primer, 3 μL of DNA template, and 9 μL of nuclease-free water. The thermocycling conditions were as follows: Initial denaturation at 95 °C for 2 minutes, 40 cycles of denaturation at 95 °C for 15 seconds, annealing at 54 °C for 15 seconds, extension at 72 °C for 15 seconds, followed by a final extension at 72 °C for 7 minutes (Bisset et al., 2014) Teladorsagia circumcincta, T. axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticei, Cooperia oncophora, Nematodirus spathiger, Chabertia ovina, and Oesophagostomum venulosum.
PCR products were visualized by electrophoresis on a 1% agarose gel stained with BioSafe™ DNA Stain. A 100 bp DNA ladder was used to estimate product size. Positive PCR products were sent to Integrated DNA Technologies (IDT), Singapore, for nucleotide sequencing.

Phylogenetic analysis
All nucleotide sequences were analyzed using MEGA11 software and BLAST. Forward and reverse sequences were aligned, trimmed, and assembled into consensus sequences. These were compared against reference ITS2 sequences in GenBank using BLAST to identify the closest matches. The selected sequences were used to construct a phylogenetic tree using the neighbor-joining method and the Kimura 2-parameter model in MEGA11. Bootstrap analysis with 1000 replicates was performed to evaluate the robustness of the tree topology (Tamura et al., 2021).

Results
Sample location and prevalence
The study was conducted at 13 deer breeding sites located across various districts in West Java Province, Indonesia. The sites included Cariu (Bogor), Panisan (Subang), Ranca Upas and Bandung Zoo (Bandung), Tahura (Bandung), Vedca (Cianjur), Pangalengan and Soreang (Bandung), Kehati and Bumi Patra (Indramayu), Gedung Negara (Cirebon), Taman Satwa (Tangerang), and Mabda Islam (Sukabumi). Figure 1 illustrates the sampling locations, indicating the spatial distribution of the deer populations across the province. 
A total of 248 deer were examined, comprising three species: A. axis (spotted deer), C. timorensis (Timor deer), and Rusa unicolor (sambar deer). Parasite infections were present in multiple locations, though the intensity of infection was consistently low (Table 1).

The prevalence of helminth eggs among all deer sampled was 18.1%. The highest infection prevalence was recorded at the Cariu site in Bogor District, where both C. timorensis and A. axis demonstrated a 60.0% positivity rate. Ranca Upas in Bandung District also showed a high prevalence in C. timorensis, with 17 out of 37 individuals (45.95%) testing positive. Moderate levels of infection were observed in Bumi Patra, Indramayu (33.33%), and Tahura, Bandung District (28.57%). In contrast, several sites, such as the Bandung Zoo, Vedca (Cianjur), and Pangalengan (Bandung), showed no positive cases. Similarly, zero prevalence was recorded in R. unicolor at Bandung Zoo. In a few sites, such as Soreang (12.5%) and Gedung Negara (20.0%), the prevalence was relatively low, affecting a minority of the sampled individuals.
All positive samples showed an EPG value of less than 50, indicating a uniformly low worm burden across all infected deer. The presence of infection was limited to light intensities and did not exceed the 50 EPG threshold in any case. This pattern was consistent across all sites where infection was detected.

PCR identification
PCR amplification targeting the ITS2 region (~370 bp) was successfully conducted on 26 larval DNA samples as shown in Figure 2.

Clear and specific bands around 370 bp were detected in samples from eight sampling sites: Tahura (lane 3), Kehati (lane 7), Bumi Patra (lane 8), Paniisan (lanes 11 and 12), Vedca (lanes 13 and 14), Cariu (lane 16), Soreang (lane 18), Mabda Islam (lane 24), and Taman Satwa (lanes 25 and 26). These results confirm the presence of S. houdemeri and/or T. axei DNA in these samples. 
In contrast, samples from five other locations—Ranca Upas (lanes 2 and 10), Pangalengan (lane 5), Soreang (lanes 6, 17–20), and Gedung Negara (lanes 9, 21–22)—did not show visible PCR bands, indicating negative results for the targeted nematodes. For Cariu, although two samples were tested (lanes 15 and 16), only one sample (lane 16) yielded a positive band, suggesting intra-location variability in parasite detection. The gel electrophoresis data align with the fecal examination findings and reinforce the molecular confirmation of gastrointestinal nematode presence in deer populations across multiple breeding locations. 

Sequencing and phylogenetic analysis
The sequencing of PCR-positive samples revealed the presence of two nematode species infecting captive deer in West Java: T. axei and S. houdemeri. BLAST comparisons against the NCBI database confirmed the identity of each sample. Samples identified as T. axei originated from Paniisan (Subang District), Vedca (Cianjur District), Taman Satwa (Tangerang District), and Mabda Islam (Sukabumi District). Meanwhile, S. houdemeri was detected in samples from Tahura (Bandung District), Kehati (Indramayu District), and Bumi Patra (Indramayu District).
The BLAST percent identity scores for T. axei ranged from 99.12% to 100%, with the highest match (100%) recorded in the sample from Paniisan (Subang District). For S. houdemeri, the percent identity ranged from 99.42% to 99.74%, with identical sequence similarity observed in samples from Kehati and Bumi Patra, both located in Indramayu District.
Further analysis was conducted using a phylogenetic tree to confirm the evolutionary relationship of the T. axei sample from Subang. As shown in Figure 3, the sample labeled “5242840 Subang 1 ITS F” clustered tightly with T.

axei isolates from Egypt, Denmark, New Zealand, and Scotland. This clade demonstrated minimal branch distances, indicating a high level of genetic similarity. The Indonesian isolate clearly grouped within the T. axei lineage and was distinctly separated from other Trichostrongylus species, such as T. vitrinus, T. retortaeformis, and T. colubriformis. H. contortus from Uganda was used as an outgroup and formed a distant branch, supporting the overall phylogenetic topology and confirming the placement of the Indonesian isolate within the T. axei clade. 
The phylogenetic analysis of Spiculopteragia spp. is presented in Figure 4.

Based on ITS region sequences, the positive samples identified from three locations—sample 3 (Tahura, Bandung District), sample 7 (Kehati, Indramayu District), and sample 8 (Bumi Patra, Indramayu District)—clustered within a monophyletic group alongside S. houdemeri sequences from Japan (LC628881.1 and AB682692.1). These sequences form a distinct clade with moderate to high bootstrap support (34–62), indicating a close evolutionary relationship among these Indonesian isolates and those from sika deer and wild ruminants in Japan.
Notably, the Indonesian samples are positioned closer to LC628881.1 (S. houdemeri from Sika deer, Japan), suggesting that the isolates found in Indonesia may share a more recent common ancestor with the East Asian strains. This close phylogenetic affinity reinforces the identification of these samples as S. houdemeri, supporting previous BLAST results with percent identity ranging from 99.42% to 99.74%. These findings indicate a potential geographical linkage or historical introduction event involving Spiculopteragia spp. among cervid hosts in East and Southeast Asia.

Discussion 
This study provides a comprehensive insight into the prevalence and molecular identification of gastrointestinal nematodes infecting captive deer in West Java, Indonesia. Through an integrative approach combining field sampling, parasitological examinations, PCR amplification, DNA sequencing, and phylogenetic analysis, two nematode species, T. axei and S. houdemeri, were successfully detected across several deer breeding centers.
The overall prevalence of gastrointestinal helminth infection was relatively low, with an average of 18.1%, and EPG values consistently below 50. This suggests a mild intensity of infection, aligning with findings from other wildlife studies indicating low parasite burdens, like in Europe (Barone et al., 2020; Brown and Morgan, 2024). Nevertheless, notable variation in prevalence was observed between breeding centers, with Cariu (Bogor District) recording the highest prevalence (60%), followed by Bumi Patra (Indramayu) and Ranca Upas (Bandung). These differences may reflect variations in management practices, environmental hygiene, enclosure density, and pasture contamination, factors previously reported as critical determinants of parasitic transmission (Kenyon et al., 2017; Vande Velde et al., 2018; Szewc et al., 2021).
Initial parasitological examination techniques revealed the presence of strongyle-type eggs. However, morphological similarities among strongyle nematode eggs limit precise identification of species (Seesao et al., 2017). Morphological identification of larvae is inherently limited in resolving species-level classification, as many closely related species share overlapping morphological traits, which can lead to less accurate results (Yan et al., 2023). Therefore, molecular identification targeting the ITS2 region of rDNA was employed, a method proven to offer high sensitivity and specificity for nematode diagnostics. The ITS2 region was selected for molecular analysis because it offers high interspecific variability with low intraspecific variation, enabling reliable species differentiation and facilitating comparisons with previous trichostrongylid studies (Roeber et al., 2013b; Sharifdini et al., 2017; Workentine et al., 2020). Electrophoresis results show successful amplification of the expected ~370 bp in samples from 9 out of 13 breeding locations.
DNA sequencing and subsequent BLAST analysis confirmed the presence of T. axei in samples from Paniisan, Vedca, Taman Satwa, and Mabda Islam, with high sequence similarity (99.12%–100%). T. axei is known for its wide host range, infecting domestic ruminants and wildlife worldwide (Halvarsson et al., 2022; Brown & Morgan, 2024). Its detection across multiple locations may indicate environmental contamination via feed or fomites and anthropogenic movement between facilities (Roeber et al., 2013a; Rinaldi et al., 2022; Mohebati et al., 2024).
Meanwhile, S. houdemeri was detected in Tahura, Kehati, and Bumi Patra centers, with BLAST identity values ranging from 99.42% to 99.74%. This parasite, typically associated with cervids, has been reported from Europe and parts of Asia (Halvarsson et al., 2022; Inoue et al., 2022). Its identification in Indonesia suggests an underrecognized geographic distribution, reinforcing the need for expanded surveillance across Southeast Asia (Nazarbeigy et al., 2021; Yan et al., 2023; Taheri et al., 2024).
The phylogenetic trees (Figure 4) further corroborated species identifications. T. axei isolates from Indonesia clustered closely with reference isolates from Japan, Iran, and the United Kingdom, consistent with previous findings highlighting the genetic conservation of this species across continents (Kumar et al., 2021). Likewise, S. houdemeri sequences grouped with those from Russia and Germany, affirming its distinct phylogenetic lineage among trichostrongyles (Sultan et al., 2014).
Although T. axei and S. houdemeri are not considered major zoonotic threats, sporadic cases of T. axei infections in humans have been reported in Mazandaran Province, Iran, particularly under conditions of poor hygiene and close animal-human interactions (Sharifdini et al., 2017). This underlines the necessity of parasite monitoring not only for wildlife health but also for minimizing potential risks to public health and livestock biosecurity (Bautista-Garfias et al., 2022).
Climate and environmental changes may also shift parasite transmission dynamics in the future. Increasing temperatures and humidity, as projected for Indonesia, could favor larval survival and enhance transmission cycles (Bautista-Garfias et al., 2022; Bhat et al., 2023; Miyankouh et al., 2025). Previous studies have shown that higher temperatures and humidity are positively correlated with the development, survival, and infectivity of gastrointestinal nematode larvae in the environment. Thus, proactive surveillance and management, including rotational grazing, strategic anthelmintic use, and improved sanitation, are recommended (Maqbool et al., 2017; Charlier et al., 2022).
Conclusion 
In conclusion, this study provides foundational data on the molecular characterization and phylogenetic relationships of T. axei and S. houdemeri in Indonesian deer. These findings contribute to the baseline knowledge of gastrointestinal nematodes in wild ruminants and highlight the importance of continuous surveillance. Future research should explore seasonal patterns, geographical coverage, and the ecological factors influencing parasite distribution to better inform wildlife health management strategies.

Ethical Considerations
Compliance with ethical guidelines
This study was conducted in accordance with ethical guidelines and approved by the Institutional Animal Ethics Committee, IPB University, Bogor, Indonesia. (Code: 252/KEH/SKE/IX/2024). Additional research permissions were obtained from each deer breeding site through official faculty recommendation letters, with site management granting approval and facilitating sample collection with animal caretakers. Fecal collection was conducted without causing harm, and examination results were shared with the management to support routine health monitoring programs.

Funding
This study was conducted with financial support from the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia, Jakarta, Indonesia (Grant No.: 22076/IT3.D10/PT.01.03/P/B/2024). 

Authors' contributions
Study design, project administration and writing: Ridi Arif; Experiments: All authors.

Conflict of interest
The authors declared no conflict of interest.


References
Arif, R., Sukmawinata, E., Nurhidayah, N., Satrija, F., Nuradji, H., & Wienanto, R., et al. (2024). Gastrointestinal nematode infections of deer and sheep in an agritourism farm in Bogor, Indonesia. Philippine Journal of Veterinary Medicine, 61(1), 59-65. [Link] 
Barone, C. D., Wit, J., Hoberg, E. P., Gilleard, J. S., & Zarlenga, D. S. (2020). Wild ruminants as reservoirs of domestic livestock gastrointestinal nematodes. Veterinary Parasitology, 279, 109041. [DOI:10.1016/j.vetpar.2020.109041] [PMID]
Bautista-Garfias, C. R., Castañeda-Ramírez, G. S., Estrada-Reyes, Z. M., Soares, F. E. F., Ventura-Cordero, J., & González-Pech, P. G., et al. (2022). A review of the impact of climate change on the epidemiology of gastrointestinal nematode infections in small ruminants and wildlife in tropical conditions. Pathogens (Basel, Switzerland), 11(2), 148. [DOI:10.3390/pathogens11020148] [PMID] 
Bhat, A. H., Tak, H., Malik, I. M., Ganai, B. A., & Zehbi, N. (2023). Trichostrongylosis: A zoonotic disease of small ruminants. Journal of Helminthology, 97, e26. [DOI:10.1017/S0022149X2300007X] [PMID]
Bisset, S. A., Knight, J. S., & Bouchet, C. L. (2014). A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples. Veterinary Parasitology, 200(1-2), 117–127. [DOI:10.1016/j.vetpar.2013.12.002] [PMID]
Brown, T. L., & Morgan, E. R. (2024). Helminth prevalence in European deer with a focus on abomasal nematodes and the influence of livestock pasture contact: A meta-analysis. Pathogens (Basel, Switzerland), 13(5), 378. [DOI:10.3390/pathogens13050378] [PMID] 
Charlier, J., Bartley, D.J., Sotiraki, S., Martinez-Valladares, M., Claerebout, E., & von Samson-Himmelstjerna, G., et al. (2022). Anthelmintic resistance in ruminants: Challenges and solutions. Advance in Parasitology, 115, 171-227. [DOI:10.1016/bs.apar.2021.12.002]
Davidson, R. K., Kutz, S. J., Madslien, K., Hoberg, E., & Handeland, K. (2014). Gastrointestinal parasites in an isolated Norwegian population of wild red deer (Cervus elaphus). Acta Veterinaria Scandinavica, 56(1), 59. [DOI:10.1186/s13028-014-0059-x] [PMID] 
Halvarsson, P., Baltrušis, P., Kjellander, P., & Höglund, J. (2022). Parasitic strongyle nemabiome communities in wild ruminants in Sweden. Parasites & Vectors, 15(1), 341. [DOI:10.1186/s13071-022-05449-7] [PMID] 
Inoue, K., Shishida, K., Kawarai, S., Takeda, S., Minami, M., & Taira, K. (2022). Helminthes detected from wild sika deer (Cervus nippon centralis) in Kanto-Chubu region, Japan. Parasitology International, 87, 102485. [DOI:10.1016/j.parint.2021.102485] [PMID]
International Union for Conservation of Nature and Natural Reserves (IUCN). 2018. The redlist of threatened species. Gland: IUCN.
International Union for Conservation of Nature and Natural Reserves (IUCN). 2025. The redlist of threatened species. Gland: IUCN. [Link]
Kenyon, F., Hutchings, F., Morgan-Davies, C., van Dijk, J., & Bartley, D. J. (2017). Worm control in livestock: Bringing science to the field. Trends in Parasitology, 33(9), 669–677.[DOI:10.1016/j.pt.2017.05.008] [PMID]
Khattak, I., Akhtar, A., Shams, S., Usman, T., Haider, J., & Nasreen, N., et al. (2023). Diversity, prevalence and risk factors associated to gastrointestinal tract parasites in wild and domestic animals from Pakistan. Parasitology International, 97, 102777. [DOI:10.1016/j.parint.2023.102777] [PMID]
Kumar, S., Gupta, S., Mohmad, A., Fular, A., Parthasarathi, B. C., & Chaubey, A. K. (2021). Molecular tools-advances, opportunities and prospects for the control of parasites of veterinary importance. International Journal of Tropical Insect Science, 41(1), 33–42. [DOI:10.1007/s42690-020-00213-9] [PMID] 
Lyons, M., Brown, T. L., Lahuerta-Marin, A., Morgan, E. R., & Airs, P. M. (2024). A molecular assessment of Ostertagia leptospicularis and Spiculopteragia asymmetrica among wild fallow deer in Northern Ireland and implications for false detection of livestock-associated species. Parasites & Vectors, 17(1), 141. [DOI:10.1186/s13071-024-06147-2] [PMID] 
Maqbool, I., Wani, Z. A., Shahardar, R. A., Allaie, I. M., & Shah, M. M. (2017). Integrated parasite management with special reference to gastro-intestinal nematodes. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, 41(1), 1–8. [DOI:10.1007/s12639-016-0765-6] [PMID] 
Meshgi, B., Jalousian, F., & Masih, Z. (2015). Phylogenetic study of haemonchus species from Iran based on morpho-molecular characterization. Iranian Journal of Parasitology, 10(2), 189–196. [PMID]
Miyankouh, R. G., Garedaghi, Y., & Amouoghli Tabrizi, B. (2025). Seasonal study of Blood Parasites: Dirofilaria immitis and dipetalonema reconditum in the guard dogs of Tabriz city, Iran. Archives of Razi Institute, 80(3), 623-627. [Link] 
Modabbernia, G., Meshgi, B., & Eslami, A. (2021). Diversity and burden of helminthiasis in wild ruminants in Iran. Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology, 45(2), 394–399. [DOI:10.1007/s12639-020-01314-5] [PMID] 
Mohebati, M., Lotfalizadeh, N., Khedri, J., Borji, H., & Ebrahimzadeh, E. (2024). An Investigation into the prevalence of gastrointestinal helminths in pigeons from Zabol, Iran. Archives of Razi Institute, 79(5), 949–954. [DOI:10.32592/ARI.2024.79.5.949] [PMID] 
Morgan, E.R., Charlier, J., Hendrickx, G., Biggeri, A., Catalan, D., & Von Samson-Himmelstjerna, G., et al. (2013). Global change and helminth infections in grazing ruminants in Europe: Impacts, trends and sustainable solutions. Agriculture, 3(3), 484-502. [DOI:10.3390/agriculture3030484]
Nazarbeigy, M., Yakhchali, M., & Pourahmad, F. (2021). Molecular study on parasitic nematodes infection in the abomasum of sheep in Ilam, Iran. Veterinary Research Forum, 12(2), 229-233. [PMID] 
Patrelle, C., Jouet, D., Lehrter, V., & Ferté, H. (2014). Development of 12 novel polymorphic microsatellite markers using a next generation sequencing approach for Spiculopteragia spiculoptera, a nematode parasite of deer. Molecular and Biochemical Parasitology, 196(2), 122–125. [DOI:10.1016/j.molbiopara.2014.09.004] [PMID]
Phetla, V., Chaisi, M., & Malatji, M. P. (2024). Epidemiology and diversity of gastrointestinal tract helminths of wild ruminants in sub-Saharan Africa: a review. Journal of Helminthology, 98, e45. [DOI:10.1017/S0022149X24000361] [PMID]
Purnama, M. T. E., Dewi, W. K., Triana, N. M., & Ooi, H. K. (2021). Serum liver enzyme profile in Timor deer (Cervus timorensis) with fascioliasis in Indonesia. Tropical Biomedicine, 38(1), 57–61. [DOI:10.47665/tb.38.1.010] [PMID]
Puspitasari, S., Farajallah, A., Sulistiawati, E., & Muladno. (2016). Effectiveness of Ivermectin and Albendazole against Haemonchus contortus in Sheep in West Java, Indonesia. Tropical Life Sciences Research, 27(1), 135–144. [PMID]
Rahman, D. A., Condro, A. A., Rianti, P., Masy’ud, B., Aulagnier, S., & Semiadi, G. (2020). Geographical analysis of the Javan deer distribution in Indonesia and priorities for landscape conservation. Journal for Natural Conservation, 54, 125795. [DOI:10.1016/j.jnc.2020.125795]
Rinaldi, L., Krücken, J., Martinez-Valladares, M., Pepe, P., Maurelli, M. P., & de Queiroz, C., et al. (2022). Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals. Advances in Parasitology, 118, 85–176. [DOI:10.1016/bs.apar.2022.07.002] [PMID]
Roeber, F., Jex, A. R., & Gasser, R. B. (2013). Impact of gastrointestinal parasitic nematodes of sheep, and the role of advanced molecular tools for exploring epidemiology and drug resistance - an Australian perspective. Parasites & Vectors, 6, 153. [DOI:10.1186/1756-3305-6-153] [PMID] 
Roeber, F., Jex, A. R., & Gasser, R. B. (2013). Next-generation molecular-diagnostic tools for gastrointestinal nematodes of livestock, with an emphasis on small ruminants: A turning point?. Advances in Parasitology, 83, 267–333. [DOI:10.1016/B978-0-12-407705-8.00004-5] [PMID] 
Rojas, A., Bass, L. G., Campos-Camacho, J., Dittel-Meza, F. A., Fonseca, C., & Huang-Qiu, Y. Y., et al. (2025). Integrative taxonomy in helminth analysis: protocols and limitations in the twenty-first century. Parasites & Vectors, 18, 87. [DOI:10.1186/s13071-025-06682-6] [PMID] 
Seesao, Y., Gay, M., Merlin, S., Viscogliosi, E., Aliouat-Denis, C. M., & Audebert, C. (2017). A review of methods for nematode identification. Journal of Microbiological Methods, 138, 37–49.[DOI:10.1016/j.mimet.2016.05.030] [PMID]
Sharifdini, M., Derakhshani, S., Alizadeh, S. A., Ghanbarzadeh, L., Mirjalali, H., & Mobedi, I., et al. (2017). Molecular identification and phylogenetic analysis of human Trichostrongylus species from an endemic area of Iran. Acta Tropica, 176, 293–299. [DOI:10.1016/j.actatropica.2017.07.001] [PMID]
Sharifdini, M., Heidari, Z., Hesari, Z., Vatandoost, S., & Kia, E. B. (2017). Molecular phylogenetics of trichostrongylus species (Nematoda: Trichostrongylidae) from Humans of Mazandaran Province, Iran. The Korean Journal of Parasitology, 55(3), 279–285. [DOI:10.3347/kjp.2017.55.3.279] [PMID] 
Sultan, K., Omar, M., Makouloutou, P., Kaneshiro, Y., Saita, E., & Yokoyama, M., et al. (2014). Molecular genetic conspecificity of Spiculopteragia houdemeri (Schwartz, 1926) and S. andreevae (Dróżdż, 1965) (Nematoda: Ostertagiinae) from wild ruminants in Japan. Journal of Helminthology, 88(1), 1–12. [DOI:10.1017/S0022149X12000521] [PMID]
Szewc, M., De Waal, T., & Zintl, A. (2021). Biological methods for the control of gastrointestinal nematodes. Veterinary Journal (London, England: 1997), 268, 105602. [DOI:10.1016/j.tvjl.2020.105602] [PMID]
Taheri, S. N., Mahmoudvand, H., Ghasemian Yadegari, J., Pourhossein, S., & Masoori, L. (2024). In vitro and in vivo effects of Astragalus Ecbatanus extract against Cutaneous Leishmaniasis. Archives of Razi Institute, 79(5), 929–934. [PMID] 
Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), 3022–3027. [DOI:10.1093/molbev/msab120] [PMID] 
Ten Doesschate, S. J., Pomroy, W. E., Tapia-Escárate, D., Scott, I., & Wilson, P. R. (2017). Establishment rate of cattle gastrointestinal nematodes in farmed red deer (Cervus elaphus). Veterinary Parasitology, 243, 105–108. [DOI:10.1016/j.vetpar.2017.06.016] [PMID]
Vande Velde, F., Charlier, J., Claerebout, E. (2018). Farmer behavior and gastrointestinal nematodes in ruminant livestock-uptake of sustainable control approaches. Frontiers in Veterinary Science, 5, 255. [DOI:10.3389/fvets.2018.00255] [PMID] 
Workentine, M. L., Chen, R., Zhu, S., Gavriliuc, S., Shaw, N., & Rijke, J., et al. (2020). A database for ITS2 sequences from nematodes. BMC Genetics, 21(1), 74. [DOI:10.1186/s12863-020-00880-0] [PMID] 
Yan, X., He, S., Liu, Y., Han, B., Zhang, N., & Deng, H., et al. (2023). Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep. Experimental Parasitology, 254, 108625.[DOI:10.1016/j.exppara.2023.108625] [PMID]
Zajac, A. M., Conboy, G.A., Little, S.E., Reichard, M.V. (2021). Veterinary clinical parasitology. New Jersey: John Wiley & Sons Inc. [Link]