Preliminary study on prevalence of hemoprotozoan parasites harbored by Stomoxys (Diptera: Muscidae) and tabanid flies (Diptera: Tabanidae) in horse farms in Nakhon Si Thammarat province, Southern Thailand

Background and Aim: Stomoxys and tabanid flies are of medical and veterinary importance because they play crucial roles in disease transmission as mechanical vectors of various hemopathogens. However, its role as a hemoprotozoan parasite vector in horse farms has not been studied. Therefore, we investigated the occurrence of hemoprotozoan parasites belonging to the genera Babesia, Theileria, and Trypanosoma in Stomoxys and tabanid flies using conventional polymerase chain reaction (PCR) and DNA sequencing. Materials and Methods: All samples (Stomoxys and tabanid flies) were collected using an Nzi trap for three consecutive days each month from November 2022 to March 2023. The flies were morphologically identified to the species level and separated according to sex. Individual (for tabanid flies) or pooled samples (consisting of three specimens of Stomoxys flies of the same species and sex collected from the same site) were used for DNA extraction. Conventional PCR was used to screen for hemoprotozoan parasite DNA, followed by Sanger sequencing to identify the species. Results: In total, 189 biting flies were collected, including four species of Stomoxys (Stomoxys bengalensis, Stomoxys calcitrans, Stomoxys indicus, and Stomoxys sitiens) and five species of tabanids (Atylotus cryptotaxis, Chrysops dispar, Tabanus megalops, Tabanus mesogaeus, and Tabanus rubidus). Stomoxys calcitrans was the most prevalent species, accounting for 58.7% (n = 111) of the collected flies. Ten (12.4%) of the 81 samples (individuals and pools) analyzed by PCR were positive for the 18S rRNA gene of the Theileria/Babesia species. Trypanosoma DNA was not detected in any sample. After performing Basic Local Alignment Search Tool searches and a phylogenetic analysis, only six samples (7.4%), including S. calcitrans (n = 2), S. sitiens (n = 2), T. megalops (n = 1), and A. cryptotaxis (n = 1), were found to be infected with Theileria sinensis. Furthermore, apicomplexan parasites, namely, Mattesia spp. and Colpodella spp., were found on S. indicus, the fungus Meira spp. was found on S. calcitrans, and the pathogenic green alga Helicosporidium spp. was found on A. cryptotaxis. Conclusion: This study is the first to report a variety of Stomoxys and tabanid flies collected from horse farms in Thailand, which were found to be infected with Theileria and Colpodella species that affect mammals, suggesting that Stomoxys and tabanid flies can be used to confirm the presence of hemoprotozoan parasites in the study area. Understanding the presence of hemoprotozoa in flies could help design vector control programs and manage various diseases in the study area.

Copyright: Phetkarl, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
In Thailand, the species diversity and seasonal abundance of Stomoxys and tabanid flies have been reported in several habitats, such as beef cattle farms, buffalo farms, and national parks [13,14]; however, little is known about the prevalence of these flies in horse farms [15].Hemoprotozoan parasites have been frequently investigated in host animals (e.g., cattle, buffaloes, goats, and horses) and in major tick vectors [9,11,16,17].Although the role of Stomoxys and tabanid flies as disease vectors for animals and humans has been studied, only a few studies have documented hemoprotozoan parasites in these flies in Thailand [18][19][20].
This study investigated the occurrence of hemoprotozoan parasites belonging to the genera Babesia, Theileria, and Trypanosoma in Stomoxys and tabanid flies in horse farms.

Ethical approval
This study was approved by the Institutional Animal Care and Use Committee of Walailak University (Approval number: WU-ACUC-65068).

Fly collection and species identification
An Nzi trap [21] was used to collect and sample adult biting flies from both farms.Flies were collected from 6 AM to 6 PM for three consecutive days each month.Flies collected each day were transported to the Parasitology Laboratory at the Akkhraratchakumari Veterinary College, Walailak University, for species identification.
In the laboratory, all collected flies were frozen for 15 min at −40°C.Only biting flies, including Stomoxys and tabanid flies, were morphologically identified to the species level using taxonomic keys [22][23][24], sexed, and counted under a stereomicroscope (Olympus, Tokyo, Japan).The identified specimens were kept individually in a 1.5 mL microcentrifuge tube containing 95% ethanol and stored at -40°C for further molecular analysis.

DNA extraction and PCR amplification
The ethanol-preserved specimens were air-dried, and genomic DNA was extracted from a combination of three body parts (the head with mouthparts, the thorax, and the abdomen) of individual specimens (tabanid flies) or pooled specimens (three specimens of Stomoxys flies) using the E.Z.N.A. ® Tissue DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions.For Stomoxys, three specimens of the same species and sex collected from the same collection site were pooled before extracting their genomic DNA.The concentration of extracted DNA was measured using a Nano-Drop™ One C Microvolume UV-visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then stored at −20°C until further analysis.
Conventional PCR was used to detect hemoprotozoan DNA in Stomoxys and tabanid flies.The primers used in this study are listed in Table-1 [25,26].Polymerase chain reactions were performed in a total volume of 12.5 µL containing 2 × 6.25 µL DreamTaq Green Master Mix (Thermo Scientific, Vilnius, Lithuania), 1-2 µL DNA template (100-200 ng/µL), 0.5 µL primers at 0.4 µM for Babesia and Theileria spp., and 1.25 µL primers at 1 µM for Trypanosoma spp.(Table -1) and nuclease-free water to a final volume of 12.5 µL.Polymerase chain reaction was performed using a Mastercycler Pro S (Eppendorf AG, Hamburg, Germany).The PCR cycle consisted of an initial denaturation step at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 30 s. Annealing temperatures were 58°C for Trypanosoma spp.and 68°C for Theileria/Babesia for 30 s.An initial extension step was performed at 72°C for 1 min, and a final extension was performed at 72°C for 5 min.For each assay, genomic DNA samples from known hemoprotozoa were used as positive controls, and nuclease-free water was used as a negative control.The PCR products were electrophoretically separated on a 1.5% agarose gel in 1× Tris-acetate EDTA buffer, stained with SERVA DNA Stain G (SERVA, Heidelberg, Germany), and visualized under UV light using the ChemiDoc™ Imaging System (Bio-Rad, Hercules, CA, USA).

DNA sequencing and phylogenetic analysis
The PCR-positive samples were purified using the E.Z.N.A. ® Cycle Pure Kit (Omega Bio-Tek) according to the manufacturer's instructions and subsequently sent to U2Bio Sequencing Service Co., Ltd.(Bangkok, Thailand) for Sanger sequencing using the same primers as those used for PCR.The obtained sequences were manually edited and assembled into complete bidirectional consensus sequences using the BioEdit software (version 7.2.5)[27].All sequencing results were compared with sequences available in the GenBank database using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov)program for species identification.Furthermore, the maximum likelihood (ML) tree was reconstructed using Tamura-Nei [28] with a gamma distribution model and tested using 1000 bootstrap replications in MEGAX software (version 10.2.6) [29] to check the genetic relatedness between Theileria spp.isolates in Thailand with those from other regions of the world and other closely related species stored in genetic databases.

Nucleotide sequence accession numbers
All sequences obtained in this study were deposited in GenBank under the following accession numbers: OQ818635-OQ818640 for Theileria sinensis, OQ818641 for Mattesia geminata, OQ818642 for Colpodella spp., OQ818643 for Meira spp., and OQ818644 for Helicosporidium spp.
The phylogenetic tree showed that the six Theileria spp.sequences were placed with the type Thung Song from Thailand (AB000270) and appeared in the same clade as T. sinensis from Malaysia and China, which was clearly separated from the other Theileria and Babesia species (Figure -1).Furthermore, the ML tree revealed that the phylogenetic positions of the H1SIF62 and H1SIF65 sequences, which were identified as M. geminata and C. tetrahymenae, respectively, from BLAST searches, fell within sister groups of the apicomplexan clade, including Theileria spp.and Babesia spp., with bootstrap values of 49% and 56%, respectively.

Discussion
To the best of our knowledge, this is the first study on the species composition of Stomoxys and tabanid flies in horse farms in Nakhon Si Thammarat Province, southern Thailand, and the detection of T. sinensis and Colpodella DNA in flies collected from horse farms.In the present study, four species of Stomoxys (S. bengalensis, S. calcitrans, S. indicus, and S. sitiens) and five tabanid fly species (A.cryptotaxis, C. dispar, T. megalops, T. mesogaeus, and T. rubidus) were identified, of which S. calcitrans was abundant.High numbers of trapped S. calcitrans, a cosmopolitan species, were similar to the findings of the previous reports from several areas of Thailand, such as zoos, dairy farms, beef cattle farms, and national parks [14,30,31].All Theileria 18S rRNA gene sequences in this study shared a high degree of similarity with Theileria spp.type Thung Song from Thailand by BLAST top hit searches (99.02%-100%); however, they formed the same cluster with T. sinensis sequences that were isolated from China and Malaysia in the phylogenetic analysis.These findings indicated that all Theileria sequences obtained in this study and Theileria spp.type Thung Song were T. sinensis.Theileria sinensis is classified as a benign Theileria species responsible for bovine theileriosis and causes benign infections in large ruminants [32].It has been recently reported in China, Malaysia, and Russia [33][34][35].In this study, T. sinensis DNA was detected in S. calcitrans, S. sitiens, A. cryptotaxis, and T. megalops.The presence of T. sinensis DNA in T. megalops is consistent with the previous studies, where T. sinensis was detected in T. megalops collected from beef cattle farms in the Nakhon Pathom province, central Thailand [19] and in bullfighting cattle from southern Thailand [11].The detection of T. sinensis DNA in Stomoxys and tabanid flies indicated the circulation and maintenance of these hemoprotozoan parasites in their respective hosts in the studied areas.
Unexpectedly, the Ba/ThF and Ba/ThR primers used in this study amplified sequences that did not closely match any characterized Theileria or Babesia species.Instead, the BLAST analyzes of the H1SIF62 and H1SIF65 sequences closely matched M. geminata (90.94% identity) and C. tetrahymenae (89.46% identity), respectively, which are members of the genus that are closely related to Apicomplexa.The ML tree confirmed the relationship between these parasites and the apicomplexan lineage, as the H1SIF62 and H1SIF65, M. geminata (AY334568), and C. tetrahymenae (MH208619) sequences fell within the sister groups of the apicomplexan clade, including Theileria spp.and Babesia spp., and were located at the base of Apicomplexa in the phylogenetic tree [36].Furthermore, these primers amplified the H1SCM34 and H2ACF19 sequences that closely matched with the M. argovae fungus (99.85% identity) and the pathogenic green alga Helicosporidium spp.(97.40% identity), respectively, according to the BLAST analyses.This suggests that these primers should be used with caution when studying the presence of Theileria and Babesia species in biting flies in the absence of sequence confirmation.
Interestingly, the DNA amplicon sequenced from S. indicus was found to have a high homology with that of C. tetrahymenae (89.46% identity), a genus closely related to the phylum Apicomplexa.Only two cases of Colpodella spp.infection in humans have recently been reported in China, suggesting that opportunistic infections with these parasites might occur in humans [37,38].Furthermore, Colpodella spp.have been identified in various mammals (e.g., cattle, Amur tigers, and horses), ticks (e.g., Rhipicephalus spp., Ixodes persulcatus, Haemaphysalis spp., and Dermacentor spp.), and water samples [39][40][41].The presence of Colpodella spp.isolated from S. indicus in this study suggests the possible prevalence of these parasites in horse farms.This is in line with the detection of Colpodella spp. in horse blood [41], which revealed that horses are at major risk for becoming a reservoir for this zoonotic parasite, Colpodella spp., and related infections in humans.Although there have been no recorded deaths in humans or animals due to these parasites, further screening of animals, ticks, and biting flies that may harbor Colpodella spp. is recommended to confirm their possible presence.
The absence of typical horse parasites, namely, Theileria equi, Babesia caballi, and Trypanosoma evansi, in the analyzed flies suggests their absence in the studied horse farms.However, this study had limitations due to the limited number of traps and collection sites, the small sample size of collected flies, and the limited sampling time.Therefore, further studies must be conducted on a larger scale by sampling more horse farms, using more traps, and having longer sampling periods.Furthermore, molecular studies targeting hemoprotozoan parasites should be performed using both horse blood and biting flies collected from farms to increase the reliability of the use of Stomoxys and tabanid flies as markers for the distribution of hemoprotozoan parasites in the study areas.

Conclusion
To the best of our knowledge, this is the first study on the species composition of Stomoxys and tabanid flies and documentation of T. sinensis DNA in S. calcitrans, S. sitiens, A. cryptotaxis, and T. megalops collected from horse farms, which causes bovine theileriosis.This is the first documented report of Colpodella spp.DNA in S. indicus.This suggests that these flies can be used as markers for monitoring the circulation of hemoprotozoan parasites in vulnerable areas, leading to effective vector control and disease management in animals.

Figure- 1 :
Figure-1: Maximum likelihood tree of 18S rRNA based on the Tamura-Nei with Gamma distribution model.Boldface letters indicate sequences acquired in this study, which include the voucher code and GenBank accession number.All reference sequences display the species name, country, and GenBank accession number.The scale bar of 0.20 indicates the evolutionary distance divergence.

Table - 4
: Results of DNA sequencing indicating the detected hemoprotozoan species in the biting flies according to collection site and sex.

Table - 3
: Prevalance of hemoprotozoa in biting flies collected from two horse farms.