Light and Scanning Electron Microscopy Studies on First-

instar of Oestrus ovis: A Maggot Causing Ophthalmomyiasis

Hazem Abdelmotaal1, Hanaa Y. Bakir2, Doaa A. Yones 2 , Rasha A. H. Attia2,

Departments of 1Ophthalmology and 2Medical Parasitology, Faculty of Medicine,

Assiut University, Egypt

ABSTRACT

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.

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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

Introduction

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

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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.

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Patient and Methods

Clinical Data:

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.

Entomologic Study:

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

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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.

Results

Macroscopic features:

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,

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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).

Discussion

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.

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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

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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

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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.

Authors' Contributions

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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28- Beaver PC: The Bot flies (Families Cuterebridae, Gastrophilidae,

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Correspondence to:Doaa A. YonesDepartment of Parasitology,Faculty of Medicine,Assiut University, Egypt.E-mail: doaayones@gmail.comShort-cut title: Light and SEM on First- instar of Oestrus ovisAuthor title: Abdelmotaal et al.

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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.

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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).

االلكتروني والماسح الضوئي بالميكروسكوب دراسات

" يرقة اوفس االوسترس لذبابة األولى اليرقة على

العيني للتدويد "مسببة

حازم المتعال عبد بكير , 1حازم يوسف يونس , 2هناء الحفيظ عبد ,2 دعاء

عطيه المنعم عبد 2رشا

الرمد الطفيليات 1قسم قسم أسيوط, , 2 و جامعة الطب كلية

جفن مقدمة: يشمل سطحي اختراق عن عبارة الخارجي العيني التدويد

الجناحين ( ). ذات يرقات الذباب بيرقات اإلنسان في العين ملتحمة و العين

( ) أكثر اآلن حتى هي األغنام في األنفية الذبابة اوفس االوسترس ويعتبر

رعاة وباألخص الريفية المناطق في العيني بالتدويد األشخاص يصيب سبب

. من مسجلة الخارجي العين تدويد حاالت من العديد وهناك والفالحين األغنام

. ومصر أوربا وجنوب إفريقيا وشمال واستراليا األوسط والشرق إفريقيا

15

: البحث للخصائص الهدف بالتفاصيل وصف هو الدراسة هذه من الهدف

الميكروسكوب باستخدام اوفس االوسترس لذبابة األولى لليرقة الشكلية

تركيب بين العالقة إليجاد وأيضا االلكتروني الماسح والميكروسكوب الضوئي

. المرض إحداث على وقدرتها اليرقات هذه

: البحث ذكر خطوات إنسان في خارجي عيني تدويد حالة الباحثون سجل

من 24عمره شديدة بأعراض أسيوط جامعة مستشفى إلى حضر عاما

. الجزء في صغيرة يرقات وجود تبين وبالفحص العين ملتحمة التهاب

. المجردة بالعين ووصفهم التقاطهم تم وقد العين ملتحمة من األمامي

الميكروسكوب و الضوئي بالميكروسكوب وتصويرهم وصفهم وكذلك

. االلكتروني الماسح

االوسترس النتائج: ذبابة يرقات أنها على اليرقات هذه على التعرف تم

بأشواك المحاطة الفم أجزاء مثل الهامة الخصائص بعض توضيح وتم اوفس

. باستخدام توضيحها تم أكثر مميزة خصائص وهناك الشكل تاجية

الجانب على متماثلين نتوءين وجود مثل االلكتروني الماسح الميكروسكوب

. الجسم أجزاء على األشواك من وصفوف األمامي

: التشخيصية الخالصة الخصائص بعض بوضوح الدراسة هذه أظهرت

والتي اوفس االوسترس لذبابة األولى لليرقات والخلفية األمامية لألجزاء

للمرض إحداثها وطريقة اليرقات هذه على التعرف سهولة في أسهمت

سرعة وأهمية مضاعفات وإحداث للداخل العين اختراقها وإمكانية

. الحاالت لهذه والعالج التشخيص

16

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