|Light and Scanning Electron Microscopy Studies on First- instar of Oestrus ovis: A Maggot Causing Ophthalmomyiasis
Hazem Abdelmotaal1, Hanaa Y. Bakir2, Doaa A. Yones2, Rasha A. H. Attia2, Departments of 1Ophthalmology and 2Medical Parasitology, Faculty of Medicine,
Assiut University, Egypt
Background: External ophthalmomyiasis refers to superficial infestation that involves the lids and conjunctiva in man by fly larvae (maggots of the order Diptera). Ophthalmomyiasis is often a benign self-limiting disease. Oestrus ovis (O. ovis) is by far the most common cause affecting mainly persons in rural areas such as shepherds and farmers. Numerous cases of external ophthalmomyiasis due to this fly were reported from Africa, Middle East, Australia, North America, Southern Europe and Egypt.
Objective: The aim of this study is to describe in details the morphological characteristics of O. ovis 1st stage larvae using light microscopy (LM) and scanning electron microscopy (SEM) and to correlate between these larval structures and their pathogenesis.
Material and methods: A 24 years-old - man presented with severe symptoms of conjunctivitis. On examination, five small sized larvae were observed in the bulbar conjunctiva. They were picked up; described and documented photographically by LM and SEM.
Results: The larvae were identified as O. ovis 1st stage larvae. They showed characteristic mouth parts, surrounded by crown shaped spines. More distinctive features revealed by SEM were mirror image papillae on the ventral side and rows of currycomb-shaped spines on the body segments.
Conclusion: This study highlighted particular diagnostic structures of the anterior and posterior ends of O. ovis 1st stage larvae, that may aid in larval identification, and explain their mechanism of pathogenesis. Most important is the early diagnosis and treatment.
KEY WORDS: Oestrus ovis, Myiasis, Ophthalmomyiasis, Egypt
Myiasis is the infestation of tissues and organs of animals or man with dipterous fly larvae for a certain period, during which they feed on the host’s dead or living tissues, liquid body-substances or ingested food. Subsequently they may cause different clinical pictures(1). Myiasis appears to be fairly common but is underestimated in many rural areas. It occurs more during warm seasons. The most common site of infestation is skin wound; less common sites are eyes, nose, paranasal sinuses, throat, and urogenital tract(2, 3).
Various species of flies are able to provoke ophthalmomyiasis, including
O. ovis (sheep nasal botfly), Fannia (latrine fly), Musca domestica (house fly), and Hypoderma (cattle botfly).(4,5). Reported predisposing factors are eye infection, young age, and debility.(6) O. ovis is by far the most common cause of ophthalmomyiasis in man.(7) Accidentally, its larvae are deposited near the eyes of human living in close proximity of live stock(8). The fly usually deposits its larvae in conjunctiva, mouth or external auditary canal(9,10). In humans, O. ovis larvae do not develop past the first instar stage.(11)
Ophthalmomyiasis due to O. ovis was described for the first time in 1947(12). More scattered cases have been reported since then from Mediterranean area like Italy, and also from Russia, Serbia (previous Yugoslavia), India, Africa, America, and Oman (13, 14). Few cases of ophthalmomyiasis were reported from Iran(15, 16). In Egypt human Ophthalmomyiasis due to O. ovis was reported by Attiah et al.(17), and Morsy and Farrag(18). Animal oestrosis was also reported by Morsy et al.(19), and Amin et al.(20)
In external ophthalmomyiasis, maggots infiltrate the conjunctiva, cornea, lacrimal ducts and eye lids(21), and present by conditions similar to viral or allergic conjunctivitis. It is characterized by pain, redness, tearing, itching and foreign body sensation in the affected eye. In rare instances, larvae may penetrate into the eye causing internal ophthalmomyiasis and optic atrophy which may lead to loss of vision(22),(23).
The aim of the present study was to describe in details the morphological characteristics of O. ovis 1st stage larvae using LM and SEM and to correlate between these larval structures and their pathogenesis.
Patient and Methods
A 24 - years - old man attended the outpatient clinic of Ophthalmology Department, Assiut University Hospital. He complained of irritation, congestion and vague feeling of foreign body crawling in his RT eye, also seen by hand mirror. He claimed being struck in his right eye by a fly during walking near a slaughterhouse. Visual acuity in this eye was 6/6, conjunctiva was congested, cornea was clear, pupil and anterior chamber were normal. Slit lamp biomicroscopy revealed five white relatively fast motile larvae. Maggots were photophobic because they avoided slit lamp beam. Two of them were removed mechanically using a sterile cotton swab after application of topical anesthetic. One larva was fixed in 4% formalin; and two larvae were fixed in 70% alcohol. Following removal of all larvae, symptoms completely resolved within few hours. Fixed larvae were referred to Parasitology Department, Faculty of Medicine, Assiut University for taxonomic identification.
Larvae preserved in 70% alcohol, were brought down to water in descending grades of alcohol 50 % and 30%, 5 minutes each. They were transferred to 5% potassium hydroxide, after puncturing the specimens on the ventral side, until soft parts were dissolved. The specimens were washed thoroughly in distilled water, dehydrated in ascending grades of alcohol 5 minutes each, cleared in clove oil for 10 minutes, mounted in Canada balsam and dried in an oven at 38°C for few days. The maggots were identified in accordance with taxonomic guide lines given by Faust et al.(24) and Peters (25).
For determination of ultrastructure, larvae were preserved in 4% formalin, washed thoroughly in distilled water, fixed in 3% glutaraldhyde and dehydrated in graded aqueous ethanol followed by critical-point drying according to Hayat (26). They were sputter-coated with gold in the sputter coating apparatus for 6 min. Specimens were examined and photographed in Scanning Electron Microscope Unit, Assiut University by JEOL-JSM-5400 LV.
Creamy white larvae, approximately one mm in length with a dark appendage at one pole that appeared as hooks by magnifying lens.
Light microscopy study:
This revealed a small fine semi translucent spindle-shaped maggot, whose body length ranged from 0.5 to 1mm and width from 0.1 to 0.25 mm. Its body consisted of eleven segments. The anterior end was equipped with two large dark oral hooks connected to the dark cephalopharyngeal skeleton. At the anterior margin of each body segment were rows of currycomb-shaped spines that continued till the caudal end (Figure1). The cephalopharyngeal skeleton showed characteristic mouth hooks (mh) that appeared as two large curved and divergent hooks. Posterior to mouth hooks were the hypopharyngeal sclerites (hps), followed by the elongated rod-shaped pharyngeal sclerites (phs). Weak body spines (ws), arranged in double rows, were confined to the anterior margin of each segment (Figure 2). There were long pointed hair-like spines (s) on the lateroventeral margins of all segments (Figure 2). Around the anus on last abdominal segment, there were claw like peri-anal spines arranged in two groups (Figure 3).
Scanning Electron microscopy study:
Ventral view of the anterior end showed a pair of strong postero-laterally directed mouth hooks with pointed distal end (arrows), and a crown of sharp pointed spines directed upward, downward and laterally(s). There were four well defined papillae, each two of appeared as mirror image of the other lateral to midline (p) (Figure 4). Top view of the maggot showed a wide mouth opening surrounded by sharp pointed rows of spines. A bridging hypopharyngeal sclerite connected the proximal end of the oral hooks. The ventral margin of the mouth opening appeared clefted (arrow) (Figure 5).
Body segments showed double rows of spines directed backward (arrows) at the anterior margin of each segment. Some spines were long, pointed, hair-like, scattered haphazardly in different directions (Figure 6). With higher magnification, the pointed end of the spines appeared either single (arrow) or bifid (arrow head) (Figure 7).
The caudal end of the maggot showed two characteristic terminal bulges carrying claw or shark teeth-shaped hooklets arranged in two groups ventrally (arrows) (Figure 8).
Three dipterous families Oestridae, Calliphoridae and Sarcophagidae are considered the main causes of myiasis in livestock and occasionally in human(27). Although myiasis in man is generally uncommon, members of the Oestridae (Diptera) may produce human myiasis in countries where the standard of hygiene is low and there is abundance of flies around the locality(12). O. ovis first stage larvae are the common cause of human myiasis in most reports where females dash to deposit their freshly hatched larvae in nose, conjunctiva, lips and mouth of the usual hosts like sheep, cattle and horse. Man serves as an accidental host(28).
In our study the patient presented with irritation, congestion and a vague feeling of foreign body crawling in his right eye.
The adult female botfly is capable of ejecting a jet of larvae, while in close proximity of the eye without striking the inner canthus. Alternatively these airborne small immature first stage larvae can be transmitted by wind for some distance(29). Larvae usually mature in the sheep nasal mucus membrane then drop to the ground and pupate.(9) The maggots are equipped with oral hooks and body spines and can burrow into the lids or penetrate the globe through the sclera into the inner structures of the eye causing internal ophthalmomyiasis. The consequent panuveitis is often severe and the visual prognosis poor(30). In very severe cases, particularly in tropical countries, the maggots eat into the tissues and convert the whole orbit into a crawling pit(22). Although the disease is self limiting, ophthalmomyiasis externa caused by O. ovis should not be regarded as a benign condition and should be treated promptly to prevent serious complication(23) . However, none of these complications were encountered in the present case (even in the follow up of the patient). It may be due to the small number of deposited larvae (only five larvae) and the short history of exposure (three days duration). The present case occurred in Summer, which is in agreement with a previous study that presented the clinical manifestations and seasonal variations of eight ophthalmomyiasis cases most of them occurring in Spring and Summer seasons(31).
Because of the serious implications in the pathogenesis of these maggots, study of their ultrastructures was done. A single superficial description of O. ovis by SEM was demonstrated by Giannetto et al.(29) and Guitton and Dorchies(31). They described the ultrastructure shape of the mouth hooks and the presence of rows of spines on the ventral and dorsal segments of the body; but they concentrated on the differentiation among 1st, 2nd and 3rd instar larvae. Ultrastructures of O. cocasicus and O. ovis instars were reported also by Guitton et al(32). The authors revised the phylogeny of family Oestridae and concluded that they were closely related morphologically. The complex and variable morphology of the attack organs of larvae explains its pathogenesis and estimates the risk of penetration of the globe(23). Our SEM examination of O. ovis 1st stage larva added more details and gave clear evidence that correlated their surface morphology with their adaptability to the host and pathogenesis. The anterior strong sharp oral hooks, the hypopharyngeal sclerite bridge connecting the proximal end of oral hooks, the clefted ventral margin of the mouth opening, the differently shaped, length, and arrangement of body spines, and characteristic terminal bulges carrying shark tooth shaped hooklets, all served the larvae to grab firmly to the conjunctiva and induce the pathogenesis of the observed signs and symptoms.
In agreement, Fathy et al (33) in their study, divided the microscopic structures of O. ovis first stage larvae into non-attachment structures that included the cephalopharyngeal skeleton and the posterior respiratory spiracles; and attachment structures consisting of mouth claws, head spines, thoracic multilayered spiny complex, thoracic and abdominal intersegmental spines and lastly the caudal spines. The authors attributed the pathogenesis induced by the O. avis 1st stage larvae to the attachment structures especially the mouth claws (or oral hooks).
The treatment of infestation is basically done by mechanical removal of the larvae by means of sterile cotton swap as described by Fathy et al (33); but they used a saline-soaked cotton swab placed across the line of larval movement, allowing the larvae to mount the swab.
In conclusion, the present case highlights two things. First, LM and SEM examination of O. ovis 1st stage larvae revealed clear description of their surface morphology, and explained their ability to attach firmly to the conjunctiva, which requires manual removal with sterile cotton swab. Second, it creates awareness among the ophthalmologists regarding larval infestation as one of the causes of conjunctivitis during the spring and summer seasons especially in developing countries like Egypt. Most important is the early diagnosis and treatment.
H Abdelmotaal, collected the samples and shared in manuscript writing; HY Bakir, wrote the manuscript and shared in laboratory work; DA Yones, reviewed the literature, shared in laboratory work and revised the manuscript; RA Attia, shared in laboratory work and revised the manuscript.
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Doaa A. Yones
Department of Parasitology,
Faculty of Medicine,
Assiut University, Egypt.
Short-cut title: Light and SEM on First- instar of Oestrus ovis
Author title: Abdelmotaal et al.
Legend of Figures of Oestrus ovis 1st stage larvae
A) Light Microscopy
Figure (1) Anterior end of larva equipped with two large dark oral hooks. Body segments showing rows of currycomb-shaped spines at the anterior margin of each segment (arrows).
Figure (2): Cephalopharyngeal skeleton showing characteristic mouth hooks (mh), hypopharyngeal sclerites (hps), elongated rod-shaped pharyngeal sclerites (phs). Body segment carrying weak body spines arranged in double rows confined to the anterior margin of each segment (ws) and hair-like spines(s) on the lateroventeral margins of all segments.
Figure (3): Last abdominal segment showing claw-like perianal spines around the anus (arrows).
B) Scanning Electron Microscopy
Figure (4): Ventral view of the anterior end showing a pair of strong laterally directed mouth hooks pointed distally (arrows); a crown of sharp pointed spines directed upward, downward and laterally(s); four well defined papillae, each two appearing as mirror images lateral to midline (p).
Figure (5): Top view with wide mouth opening surrounded by sharp pointed rows of spines. Hypopharyngeal sclerite appeared as a bridge connecting the proximal end of oral hooks. The ventral margin of the mouth opening is clefted (arrow).
Figure (6): Body segments with double rows of spines at the anterior margin of each segment and directed backward (arrows), some spines were long, pointed hair-like scattered haphazardly in different directions.
Figure (7): Higher magnification of the pointed end of the spines that appear either simple (arrow) or bifid (arrow head).
Figure (8): Caudal end with two characteristic terminal bulges carrying claw or shark teeth shaped hooklets arranged in two groups (arrows).
دراسات بالميكروسكوب الضوئي والماسح الالكتروني على اليرقة الأولى لذبابة الاوسترس اوفس"يرقة مسببة للتدويد العيني"
حازم عبد المتعال حازم1 , هناء يوسف بكير2 , دعاء عبد الحفيظ يونس 2 , رشا عبد المنعم عطيه2
قسم الرمد 1 و قسم الطفيليات 2, كلية الطب , جامعة أسيوط
مقدمة: التدويد العيني الخارجي عبارة عن اختراق سطحي يشمل جفن العين و ملتحمة العين في الإنسان بيرقات الذباب ( يرقات ذات الجناحين ). ويعتبر الاوسترس اوفس ( الذبابة الأنفية في الأغنام ) هي حتى الآن أكثر سبب يصيب الأشخاص بالتدويد العيني في المناطق الريفية وبالأخص رعاة الأغنام والفلاحين.وهناك العديد من حالات تدويد العين الخارجي مسجلة من إفريقيا والشرق الأوسط واستراليا وشمال إفريقيا وجنوب أوربا ومصر.
الهدف البحث: الهدف من هذه الدراسة هو وصف بالتفاصيل للخصائص الشكلية لليرقة الأولى لذبابة الاوسترس اوفس باستخدام الميكروسكوب الضوئي والميكروسكوب الماسح الالكتروني وأيضا لإيجاد العلاقة بين تركيب هذه اليرقات وقدرتها على إحداث المرض.
خطوات البحث: سجل الباحثون حالة تدويد عيني خارجي في إنسان ذكر عمره 24 عاما حضر إلى مستشفى جامعة أسيوط بأعراض شديدة من التهاب ملتحمة العين. وبالفحص تبين وجود يرقات صغيرة في الجزء الأمامي من ملتحمة العين. وقد تم التقاطهم ووصفهم بالعين المجردة وكذلك وصفهم وتصويرهم بالميكروسكوب الضوئي و الميكروسكوب الماسح الالكتروني.
النتائج: تم التعرف على هذه اليرقات على أنها يرقات ذبابة الاوسترس اوفس وتم توضيح بعض الخصائص الهامة مثل أجزاء الفم المحاطة بأشواك تاجية الشكل . وهناك خصائص مميزة أكثر تم توضيحها باستخدام الميكروسكوب الماسح الالكتروني مثل وجود نتوءين متماثلين على الجانب الأمامي وصفوف من الأشواك على أجزاء الجسم.
الخلاصة: أظهرت هذه الدراسة بوضوح بعض الخصائص التشخيصية للأجزاء الأمامية والخلفية لليرقات الأولى لذبابة الاوسترس اوفس والتي أسهمت في سهولة التعرف على هذه اليرقات وطريقة إحداثها للمرض وإمكانية اختراقها العين للداخل وإحداث مضاعفات وأهمية سرعة التشخيص والعلاج لهذه الحالات.