J Arthropod-Borne Dis, September 2017, 11(3): 441–445 SM Hoseini et al.: Histopathological Study of … 441 http://jad.tums.ac.ir Published Online: September 08, 2017 Case Report Histopathological Study of Esophageal Infection with Gasterophilus pecorum (Diptera: Oestridae) in Persian Onager (Equus hemionus onager) *Seyed Mohammad Hoseini 1, Bahram Ali Zaheri 2, Mohamad Ali Adibi 2, Hooman Ronaghi 3, Amir Hossein Moshrefi 4 1Department of Pathobiology, Babol Branch, Islamic Azad University, Babol, Iran 2Department of Environmental Protection Semnan, Semnan, Iran 3Department of Microbiology, Faculty of Specialized Veterinary Science, Science and Research Branch, Islamic Azad University, Tehran, Iran 4Young Researchers and Elite Club, Babol Branch, Islamic Azad University, Babol, Iran (Received 2 Nov 2016; accepted 22 July 2017) Abstract Background: The larval stages of Gasterophilus are obligate parasites in the gastrointestinal tract of equine account- able for pathologic ulcers in the Persian onager gastrointestinal. The aim of the current report was to study the histo- pathological change with G. pecorum larvae in the esophagus of a Persian onager. Methods: This study was performed in Iranian Zebra propagation and breeding site in Khartouran National Park, southeast of Shahrud City, Semnan Province, Iran in 2014. Following a necropsy with specific refer to esophagus of one adult female Persian onager were transmitted to the laboratory. After autopsy, parasites collected from the esophagus were transmitted into 70% alcohol. For histopathological investigation, tissue samples were collected from the esophagus. The tissues were fixed in 10% buffered formalin, and conformity routine processing, there were stained with Hematoxylin and eosin. Results: After clarity by lactophenol parasites were identified as G. pecorum. Microscopic recognition contained hyperemia, inflammatory cell infiltration, epithelial destruction, esophageal gland hyperplasia. Conclusion: This is the first survey of G. pecorum and histopathological study in the Persian onager esophagus in the world. Keywords: Persian onager, Gasterophilus pecorum, Epithelial destruction Introduction The Persian onager (Equus hemionus on- ager), a wild donkey endemic to Iran, is clas- sified as critically endangered on the Interna- tional Union for Conservation of Nature Red List. The Asian wild donkeys were confined in successive periods but ecology of the two residual crowds, determined in preserved re- gion in Touran National Park and Bahram-e- Goor Reserve (1). The genus Gasterophilus (Diptera: Oes- tridae) contains nine species. Equids are hosts to the larvae of the Gasterophilus type caus- ing gastrointestinal myiasis. Gasterophilus is specified by dysphasia, gastrointestinal ulcer ations, intestinal obstruction or volvulus, rec- tal prolapses, anemia, diarrhea and digestive disturbances. The adult flies are not parasitic and are large, 11–15mm in length. Adult Gasteroph- ilus spp. flies lay their eggs to host hairs. G. pecorum is an exception as females lay their eggs in brown-haired person, leaves, and stalks of plants (2, 3). After hatching, the larvae tunnels into the tissue of the host, larvae at the first stage at- tain the oral cavity of equine passively (G. intestinalis, G. pecorum) or actively, the first stage larvae hatch and moult to L2, which *Corresponding author: Dr Seyed Mohammad Hosseini, E-mail: dr_hosseini2323@yahoo.com https://en.wikipedia.org/wiki/Fly https://en.wikipedia.org/wiki/Oestridae mailto:dr_hosseini2323@yahoo.com J Arthropod-Borne Dis, September 2017, 11(3): 441–445 SM Hoseini et al.: Histopathological Study of … 442 http://jad.tums.ac.ir Published Online: September 08, 2017 can be available in various regions of the gas- trointestinal tract, and in L3 remains depend- ent to the mucosa for 8–10 months (4, 5). Gasterophilus pecorum, G. inermis, and G. haemorrhoidalis are just reported in finite regions of Europe and Eastern Countries (6). The damage the bot fly reasons happens after the larvae arrive the animal's mouth and gastrointestinal tract. When the first instar lar- vae tunnel into the mouth, the horse may ex- perience intense inflammation, as well as the expansion of pus pockets and loosened teeth. Loss of appetite may develop due to the lar- va's resident. As the second and third instar larvae reside the gastrointestinal tract and bind to the stomach and intestine, variable compli- cations can occur. Severe infestation of these larvae can cause anemia, esophageal paraly- sis, ulcerated stomach, chronic gastritis, stom- ach rupture and squamous cell tumors (7). Esophageal disorders, exception obstruc- tion is not common to observe in equine. Lit- tle is known about the parasite spectrum of this species. Accordingly, there was severity of the infection in this area of robot flies. The aim of the current report was to study the histopathological change with G. pecorum larvae in the esophagus of a Persian onager. Materials and Methods This study was performed in Iranian Zebra propagation and breeding site in Khartouran National Park, southeast of Shahrud City, Semnan Province, Iran (latitude 36.736536, longitude 55.700684) in 2014, with a tem- perate climate. A cervical vertebral fracture after collision with a fence was diagnosed as cause of death of the 20-yr-old female Per- sian onager. Probably it escaped from some- thing and did not see a fence and subse- quently broken neck lead to death. Following a field, necropsy gastrointesti- nal system was attentively removed and trans- ferred directly to the Laboratory of Veteri- nary Diagnostic Medicine of the Islamic Azad University-Babol Branch for histopathologic and parasitological examination. The esophagus was assayed for parasite infections. The large changes were recorded, and myiasis was collected and transferred into 70% alcohol (Jahan Alcohol Teb Co., Arak 454546, Iran). The parasites were detected as G. pecorum by light microscope with referral to key Zumpt keys (8) using stereo micro- scopes with 10X to 40X magnification. Tis- sue samples and myiasis were used for his- topathological examinations fix the tissue im- mediately in 10% buffered formalin, paraffin- embedded and sections were cut using a ro- tary microtome (Leitz, 1512, Germany) at 5μm and stained with hematoxylin and eosin (H and E). The study was approved by the Animal Ethics Committee of Islamic Azad Universi- ty, Babol Branch, Babol, Iran. Results Esophageal necropsy revealed that the onager was infected to myiasis. Totally, 87 third larval stage of G. pecorum were re- moved from esophageal tissue of the animal (Fig. 1). The third larval stage of G. pecorum was the arrangement of denticles on the pseudo- cephalon into 3 groups, 2 lying laterally and a third centrally in front of the mouth hooks (Fig. 2). We found gastric myiasis caused by G. pecorum (365 larvae). The histopathological study revealed different part of myiasis (Fig. 3). Microscopic examination showed epithe- lial destruction, esophageal gland hyperplas- ia, hyperemia, lymphocyte and macrophage infiltration in mucosa and submucosa of esophagus of the Equus hemionus infected by Gasterophilus (Fig. 4). J Arthropod-Borne Dis, September 2017, 11(3): 441–445 SM Hoseini et al.: Histopathological Study of … 443 http://jad.tums.ac.ir Published Online: September 08, 2017 Fig. 1. Third larval stage of Gasrophilus pecorum A. Ventral view of pseudocephalon B. ventral view C. dorsal view Fig. 2. Gasterphilus larvae on the esophageal muco- sal membrane of an onager Fig. 3. Cross-sections Gasterphilus larvae (10x), H and E Fig. 4. Cross sections of intraluminal myiasis, Inflammatory cell infiltration (1), epithelial destruction (2), Esopha- geal gland hyperplasia (3). H and E. x40 Discussion Botfly infestation has been reported in dif- ferent countries can cause economic losses in domestic animals. The presence of Gaster- ophilus species has been much studied in Asia extensively as the most pathogenic Gaster- ophilus species on horses. Accomplished investigations in different parts of the world, incidence ranged from 11% B J Arthropod-Borne Dis, September 2017, 11(3): 441–445 SM Hoseini et al.: Histopathological Study of … 444 http://jad.tums.ac.ir Published Online: September 08, 2017 to 100% including 11.1% in Israel (9), 12.3% in Sweden (10), 43% in Ireland (11), 34% in France (12), 53% in England and Wales (13), 58% in Belgium (14), 82.2% in Italy (6), 65% in Switzerland (15), 98.7% in Kentucky, USA )16) and 100% in Morocco (17, 18). Bots in the alimentary tract were identi- fied as third stage larvae associated with ul- cers include G. pecorum, G. nigricornis and G. nasalis (19). Gasterophilus pecorum has been studied in Asia where it is regarded as the most pathogenic Gasterophilus species on horses (8). A study in China was done on the diag- nosis of the larval Gasterophilus species in 90 equines, from 2008 to 2013 revealed the all-90 (100%) equines were infested via lar- val Gasterophilus, and 3723 secondary instar larvae (L2) as well as 63778 third instar lar- vae (L3). Six types of Gasterophilus were recognized include G. pecorum 88.94%, G. nigricornis 4.94%, G. nasalis 3.93%, G. haem- orrhoidalis 1.91%, G. intestinalis 0.19%, and G. inermis 0.087% (20). In Iran, the onager was infected by myiasis (G. pecorum) and nem- atode (Habronema muscae) (21). According to our results, G. pecorum is more adaptable to the local environment in Khartouran National Park. The association with this unique comportment and the desert steppe ecosystem can help describe the situation. Water availability limits the activity area of wild animals in a region such as Khar- touran National Park, which has high evapo- ration, limited surface runoff, and low pre- cipitation. A study in Kalamaili showed that the oviposition sites of G. pecorum were of- ten near a water source (3). Frequent drink- ing at water sources may increase the risk of G. pecorum infection. Thus, the equids in arid desert grasslands have a higher intensity of Gasterophilus spp. Conclusion This is the first report of this parasite and histopathological study in the Persian onager esophagus in the world. Acknowledgements The authors sincerely want to thank the people contributed for the completion of this research project, especially thanks to Ms Es- lami, because of her helps in laboratory ex- periments. The authors declare that there is no conflict of interest. References 1. Tatin L, Darreh-Shoori BF, Tourenq C, Tatin D, Azmayesh B (2003) The last populations of the Critically Endangered onager Equus hemionus onager in Iran: urgent requirements for protection and study. Oryx. 37(4): 488–491. 2. Gökçen A, Sevgili M, Altaş MG, Camk- erten İ (2008) Presence of Gasterophi- lus species in Arabian horses in San- liurfa region. Turkiye Parazitol Derg. 32: 337–339. 3. Liu SH, Hu DF, Li K (2015) Oviposition site selection by Gasterophilus pecorum (Diptera: Gasterophilidae) in its habitat in Kalamaili Nature Reserve, Xinjiang, China. Parasite. 22: 34. 4. Studzińska MB, Wojcieszak K (2009) Gas- terophilus sp. botfly larvae in horses from the south-eastern part of Poland. Bull Vet Inst Pulawy. 53: 651–655. 5. Ganjali M, Keighobadi M (2016) A Rare Case of Gastric Myiasis in a Lion Caused by Gasterophilus intestinalis (Diptera: Gasterophilidae)-Case Report. J Arthro- pod Borne Dis. 10(3): 423–425. 6. Otranto D, Milillo P, Capelli G, Colwell DD (2005) Species composition of Gaster- ophilus spp. (Diptera, Oestridae) causing equine gastric myiasis in southern Italy: parasite biodiversity and risks for extinc- tion. Vet Parasitol. 133(1): 111–118. J Arthropod-Borne Dis, September 2017, 11(3): 441–445 SM Hoseini et al.: Histopathological Study of … 445 http://jad.tums.ac.ir Published Online: September 08, 2017 7. Mashayekhi M, Ashtari B (2013) Study of Gasterophillus role in equine gastric ulcer syndrome in Tabriz area. Bull Env Pharmacol Life Sci. 2(12): 169–172. 8. Zumpt F (1965) Myiasis in man and ani- mals in the Old World. A textbook for physicians, veterinarians and zoologists, Butterworths, London. 9. Sharir B, Pipano E, Markovics A, Danieli Y (1987) Field studies on gastrointesti- nal infestation in Israeli Horses. Isr J Vet Med. 43: 223–227. 10. Hoglund J, Ljungstrom BL, Nilsson O, Lundguist H, Osterman E, Uggla A (1997) Occurrence of Gasterophilus in- testinalis and some parasitic nematodes of horses in Sweden. Acta Vet Scand. 38: 157–165. 11. Sweeney HJ (1990) The prevalence and pathogenicity of Gasterophilus intesti- nalis larvae in horses in Ireland. Irish Vet J. 43: 67–73. 12. Bernard N, Collobert C, Tariel G, Lamidey C (1994) Epidemiological survey of bot infection in horses at necropsy in Normandy from April 1990 to March 1992. Rec Med Vet. 170: 231–235. 13. Edwards GT (1982) The prevalence of Gasterophilus intestinalis in horses in northern England and Wales. Vet Para- sitol, 11: 215–222. 14. Agneessens J, Engelen S, Debever P, Vercruysse J (1998) Gasterophilus in- testinalis infections in horses in Bel- gium. Vet Parasitol. 77: 199–204. 15. Brocard P, Pfister K (1991) The epidemi- ology of gasterophilosis of horses in Switzerland. Schweiz Arch Tierheilkd. 133: 409–416. 16. Drudge JH, Lyons ET, Wyant ZN, Tol- liver SC (1975) Occurrence of second and third instars of Gasterophilus intes- tinalis and Gasterophilus nasalis in stomachs of horses in Kentucky. Am J Vet Res. 36: 1585–1588. 17. Pandey VS, Ouhelli H, Elkhalfane A (1980) Observations on the epizootiol- ogy of Gasterophilus intestinalis and G. nasalis in horse in Morocco. Vet Para- sitol. 7: 347–356. 18. Tavassoli M, Bakht M (2012) Gastrophi- lus spp. myiasis in Iranian equine. Sci Parasitol. 13(2): 83–86. 19. Liu SH, Hu DF, Li K (2016a) Parasites observed in the proximal alimentary tract of a Przewalski's horse in China. Equine Veterinary Education. Availa- ble at: http://onlinelibrary.wiley.com/doi/ 10.1111/eve.12593/full 20. Liu SH, Li K, Hu DF (2016b) The inci- dence and species composition of Gas- terophilus (Diptera, Gasterophilidae) caus- ing equine myiasis in northern Xin- jiang, China. Vet Parasitol. 217: 36–38. 21. Zaheri BA, Ronaghi H, Youssefi MR, Hoseini SM, Omidzahir S, Dozouri R, Eshkevari SR, Mousapour A (2015) Gas- terophilus pecorum and Habronema mus- cae in Persian onager (Equus hemionus onager), histopathology and parasitology survey. Comp Clin Path. 24(5): 1009– 1013. http://onlinelibrary.wiley.com/doi/