First Reported Case of Accidental Gastric Myiasis Caused by 
Myiasis is the infestation of living vertebrates by larvae (“maggots”) of dipterous flies. Depending on the host-parasite relationship, myiasis is classified as obligatory, facultative or accidental. The obligatory myiasis-causing fly species require a living host for their life cycle. Their larvae develop inside the host, feed on its tissues and can remain there for months. In facultative myiasis, the fly larvae can develop in either dead or living organic material. Accidental myiasis occurs when fly eggs or larvae are accidentally ingested with food or water and pass through the digestive tract . The first group includes the Gasterophilus species (Diptera: Oestridae), whose larvae are endoparasites of domestic horses, donkeys and wild equids [ – ]. During the summer months, adult females of Gasterophilus spp. lay eggs on host hairs or, in the case of Gasterophilus pecorum , on plants. After licking or oral ingestion of the eggs, the first instar larvae hatch. Depending on the species, they penetrate the host’s skin or oral mucosa to moult into second instar larvae. Third instar larvae remain in specific areas of the gastrointestinal tract for many months, e.g. Gasterophilus haemorrhoidalis , Gasterophilus intestinalis and G. pecorum in the stomach. They are eventually excreted in the faeces and pupate in manure or soil to start the next generation of adult flies . Although the attachment of their second and third instar larvae with the cephalopharyngeal hooks causes ulcerative lesions in the gastrointestinal mucosa, infestation in horses usually remains subclinical . Non-equids are unsuitable hosts for Gasterophilus spp . Penetration of first instar Gasterophilus larvae into the skin or oral mucosa of non-equids results in ophthalmomyiasis or transient local (muco)cutaneous myiasis. Such cases have been reported in humans [ – ]. Occasionally, Gasterophilus spp. larvae, which cause accidental myiasis, have been found in the digestive tracts of carnivores and scavengers, including dogs [ – ], hyenas , raccoon dogs , lions [ – ], badger  and vultures . Here we report the first case of accidental gastric myiasis caused by Gasterophilus larvae in a wolf. Case Presentation In February 2023, a gray wolf ( Canis lupus ) was hunted near a settlement in the Kostanay region (northern Kazakhstan). Its gastrointestinal tract was frozen for later examination. The thawed stomach, which was otherwise empty, contained four cylindrical, cream-coloured maggots, approximately 15 mm long, attached to the gastric mucosa. The maggots were washed in physiological saline and morphologically identified using the key of Zumpt , three as second instar larvae and one as third instar larva (Fig. ) of a Gasterophilus sp. For molecular species identification , genomic DNA was extracted from the lateral part of each larva, which has no morphological value for identification, using phenol-chloroform-isoamyl alcohol (PCI): First, 200 µl lysis buffer and 5 µl proteinase K were added, then PCI was added to the solution and centrifuged at 15,000 rpm at 25ºC for 10 min. The supernatant was precipitated with absolute ethanol (1:4) in a new tube at − 20ºC for 24 h. The liquid was then centrifuged again at 15,000 rpm at 4ºC for 10 min. The sediment was air dried and TE (100 mM Tris and 25 mM EDTA, pH = 8.0) was added to the sediment and stored at − 20 °C until analysis. The mitochondrial cytochrome c oxidase subunit 2 ( cox 2) gene was amplified by polymerase chain reaction (PCR) using the following primer pair: TL2-J-3037 (5′-ATGGCAGATTAGTGCAATGG-3′, forward) and TK-N-3785 (5′- GTTTAAGAGACCAGTACTTG − 3′, reverse) . The total reaction volume was 25 µl: 12.5 µl DreamTaq MasterMix (ThermoFisher Scientific, K1071, Lithuania), 1 µl of each primer, 1 µl of template DNA and 9.5 µl ddH2O. The PCR programme consisted of denaturation at 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s with final polymerization at 72 °C for 5 min. Amplicons were analysed by 1% agarose gel electrophoresis, purified using ExoSAP-IT (USB, Cleveland, USA) and sequenced by standard Sanger sequencing on an Applied Biosystems 3130xl automated genetic analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, USA). The nucleotide sequence was analysed using MUSCLE (Multiple Sequence Comparison by Log-Expectation) software for multiple sequence alignment. Evolutionary analysis was performed by maximum likelihood and the Tamura-Nei model using MEGA11 software . The nucleotide sequence was compared with sequences of other Gasterophilus spp. in the GenBank database using the BLAST algorithm ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). A 704 bp amplicon was obtained from the maggot and the nucleotide sequence has been deposited in the NCBI GenBank under accession no. PP584667.1. The maggot was identified as that of G. pecorum using the primer pair on the mitochondrial cox2 region and by comparing the nucleotide sequences obtained with references from the GenBank database. Phylogenetic analysis showed that the sampled G. pecorum , together with reference samples of the same species, belonged to a single clade (Fig. ). 

In February 2023, a gray wolf ( Canis lupus ) was hunted near a settlement in the Kostanay region (northern Kazakhstan). Its gastrointestinal tract was frozen for later examination. The thawed stomach, which was otherwise empty, contained four cylindrical, cream-coloured maggots, approximately 15 mm long, attached to the gastric mucosa. The maggots were washed in physiological saline and morphologically identified using the key of Zumpt , three as second instar larvae and one as third instar larva (Fig. ) of a Gasterophilus sp. For molecular species identification , genomic DNA was extracted from the lateral part of each larva, which has no morphological value for identification, using phenol-chloroform-isoamyl alcohol (PCI): First, 200 µl lysis buffer and 5 µl proteinase K were added, then PCI was added to the solution and centrifuged at 15,000 rpm at 25ºC for 10 min. The supernatant was precipitated with absolute ethanol (1:4) in a new tube at − 20ºC for 24 h. The liquid was then centrifuged again at 15,000 rpm at 4ºC for 10 min. The sediment was air dried and TE (100 mM Tris and 25 mM EDTA, pH = 8.0) was added to the sediment and stored at − 20 °C until analysis. The mitochondrial cytochrome c oxidase subunit 2 ( cox 2) gene was amplified by polymerase chain reaction (PCR) using the following primer pair: TL2-J-3037 (5′-ATGGCAGATTAGTGCAATGG-3′, forward) and TK-N-3785 (5′- GTTTAAGAGACCAGTACTTG − 3′, reverse) . The total reaction volume was 25 µl: 12.5 µl DreamTaq MasterMix (ThermoFisher Scientific, K1071, Lithuania), 1 µl of each primer, 1 µl of template DNA and 9.5 µl ddH2O. The PCR programme consisted of denaturation at 94 °C for 2 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s with final polymerization at 72 °C for 5 min. Amplicons were analysed by 1% agarose gel electrophoresis, purified using ExoSAP-IT (USB, Cleveland, USA) and sequenced by standard Sanger sequencing on an Applied Biosystems 3130xl automated genetic analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Carlsbad, USA). The nucleotide sequence was analysed using MUSCLE (Multiple Sequence Comparison by Log-Expectation) software for multiple sequence alignment. Evolutionary analysis was performed by maximum likelihood and the Tamura-Nei model using MEGA11 software . The nucleotide sequence was compared with sequences of other Gasterophilus spp. in the GenBank database using the BLAST algorithm ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ). A 704 bp amplicon was obtained from the maggot and the nucleotide sequence has been deposited in the NCBI GenBank under accession no. PP584667.1. The maggot was identified as that of G. pecorum using the primer pair on the mitochondrial cox2 region and by comparing the nucleotide sequences obtained with references from the GenBank database. Phylogenetic analysis showed that the sampled G. pecorum , together with reference samples of the same species, belonged to a single clade (Fig. ). 

In general, fly larvae accidentally ingested by carnivores in food or water pass directly through the gastrointestinal tract and are excreted in the faeces, either alive or dead. Examples of such events in canids include maggots of Cochliomyia hominivorax , Lucilia sericata (both family Calliphoridae) , Sarcophaga sp. (Sarcophagidae) , Dermatobia hominis , Cuterebra sp. (both Oestridae) , Hermetia illucens (Stratiomyidae)  and Eristalis tenax (Syrphidae) . In this case, however, second and third instar Gasterophilus larvae were found attached to the wall of the empty stomach. This suggests that the wolf either killed an infested horse or found it dead and ingested its intestines. Alternatively, the wolf could have ingested the discarded entrails of a slaughtered horse, but this is less likely as the Kazakh people commonly use and consume the gastrointestinal tract of horses. More than half a day, if not several days, must have elapsed between the ingestion of the Gasterophilus larvae and the killing of the wolf, as the total gastric emptying time in canids is estimated to be around 20 h . As wolves devour their prey quickly and in great competition , the Gasterophilus larvae were able to survive the ingestion process intact; they attached themselves to the stomach wall with their cephalopharyngeal hooks and remained there for a prolonged period of time. This is supported by similar findings in lions [ – ] and vultures , as well as by experimental feeding studies in dogs: third instar Gasterophilus larvae fed to dogs were found attached to the stomach wall several days after ingestion . Although carnivores are considered dead-end hosts for Gasterophilus spp. and do not contribute to their life cycle , such cases meet the definition of accidental myiasis by Scholl et al. . This is the first reported case of accidental gastric myiasis caused by Gasterophilus larvae in a wolf. The species was molecularly identified as G. pecorum , a common species in equids in Kazakhstan and other Asian countries [ – ] and well adapted to cold temperatures . Kazakhstan has the third largest wolf population in the world (estimated at 30,000) . Wolves are synanthropic and also prey on domestic ungulates, including horses, particularly in Asian regions where livestock farming is widespread and wild prey has been depleted . Traditionally kept for meat or milk, most horses in Kazakhstan roam the steppes, often all year round , making them easy prey. Virtually all horses in this country are infested with Gasterophilus spp. larvae throughout the year . It is therefore to be expected that cases such as the present one will occur sporadically. 

