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Masters Theses 1911 - February 2014
1979
Studies on the interactions of Thelazia sp., introduced eyeworm parasites of cattle, with their definitive and intermediate hosts in Massachusetts.
Christopher John Geden University of Massachusetts Amherst
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STUDIES ON THE INTERACTIONS OF THELAZIA SP. , INTRODUCED
EYEWORM PARASITES OF CATTLE, WITH THEIR DEFINITIVE AND
INTERMEDIATE HOSTS IN MASSACHUSETTS
A Thesis Presented
By
CHRISTOPHER JOHN GEDEN
Submitted to the Graduate School of the University of Massachusetts in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
September 1979
Entomology STUDIES ON THE INTERACTIONS OF THELAZIA SP. , INTRODUCED
EYEWORM PARASITES OF CATTLE, WITH THEIR DEFINITIVE AND
INTERMEDIATE HOSTS IN MASSACHUSETTS
A Thesis Presented
By
CHRISTOPHER JOHN GEDEN
Approved as to style and content by*
6 v- a (Dr. John G. Stoffolano, Jr.), Chairperson of Committee
(Dr, John D, Edman), Member /
qU± ~ ft ^ (Dr, Chih-Ming Yin), Member'
ii DEDICATION
To my parents, George F. and Doris L. Geden, for their many years of encouragement, love, and emotional support.
iii ACKNOWLEDGEMENT
I would like to express my sincere appreciation to my advisor,
Dr. John G. Stoffolano, Jr., for his constant support and encouragement throughout the course of this study. His insights and suggestions were invaluable assets towards its completion, as was his careful reading of the manuscript. I would also like to thank Dr, Stoffolano for allowing me a considerable degree of flexibility and independence in deciding the directions that the project would take and for the amiability and open¬ ness which characterize the deep concern which he shows for his students.
I also wish to thank the other members of my committee, Dr. John D.
Edman and Dr. Chih-Ming Yin, for their advice and careful reading of the manuscript,
My gratitude is also extended to Ms. Mary Wariner and to Messrs.
Robert Jones and Norman Lapolice for assisting in the many hours of often tedious, and occasionally vile, work which went into this study.
Also to Mr. Gordon Cook of Hadley for allowing me to make frequent trips to his dairy farm to collect breeding material for the fly colony.
A special note of thanks is extended to my mother for her many hours at the typewriter during these past few months.
Finally, I wish to thank all of the faculty, staff, and graduate students of the Department of Entomology for making my degree program the enjoyable and rewarding experience which it has been.
iv ABSTRACT
A survey (1976) in Massachusetts of 18 face fly populations total¬ ing 5,479 flies showed all samples to include at least some individuals parasitized by immatures of the nematode genus Thelazia. Parasitism in the flies ranged from 0.4 to 13.2%. In 1977* a survey of face fly popu¬ lations associated with 3 separate dairy herds in the western part of the state revealed a bimodal pattern of parasitization. Two peaks, one in early summer and one in late summer, were observed. Dissections of field-collected face flies (Musca autumnalis) and house flies (M^ domes- tica) in 1978 indicated the unsuitability of the latter as an intermedi¬ ate host. Of 1,635 flies of both species examined, 5.7% of the face flies were parasitized by Thelazia larvae while none of the house flies con¬ tained any viable nematodes. Two house flies were found with dead, mela- nized parasitic nematodes in the abdomen indicative of a host response.
A survey for Thelazia sp. in Massachusetts bovines was conducted in
1977-1978. Two species of eyeworms, Thelazia gulosa and T\_ skrjabini, were recovered from cattle eyes obtained from abattoirs. Of 795 eyes
examined 97 (12.2%) were infected with Thelazia sp. Seventy-four eyes were parasitized by gulosa and 8 by T. skr jabini alone, with 15 show¬
ing mixed infections. Infections were almost entirely subclinical on post-mortem examination; however, in one eye a nodule of gulosa worms
just under the surface of the conjunctiva was associated with extensive lesions and general conjunctivitis. In another instance, a mass of juve¬ nile T. gulosa parasites in the conjunctival sac appeared to have resul¬ ted in slight scarification, localized edema and redness.
v Development of the eyeworm Thelazia gulosa was observed in experi¬ mentally infected female face flies (Husca autumnalls). Following remo¬ val from bovine eyes obtained from local abattoirs, adult nematodes were dissected and larval parasites fed to restrained 5-7 day old flies which had been starved for 12 hr. Larvae penetrated the mid-gut of the flies
1-4 hr post-infection. On day 3, all live larvae were found in capsules attached to the abdominal body wall. No development was observed in un¬ encapsulated larvae. Development to the invasive third instar larva was completed by day 9. Measurements of parasites on days 3> 6 and 9 post¬ infection are presented.
vi TABLE OF CONTENTS
DEDICATION. i±i
ACKNOWLEDGEMENT Iv
ABSTRACT. v
LIST OF TABLES. i*
LIST OF FIGURES. x
Chapter I, LITERATURE REVIEW. 1
The Face Fly, Musca autumnal Is DeGeer. 1 The Genus Thelazia Bose. 6 Thelaziasis in North America.. 8 Vector-Parasite Interactions. 12 Pathology, Diagnosis and Treatment of Bovine Thelaziasis. 19
II. GEOGRAPHIC RANGE AND TEMPORAL PATTERNS OF PARASITIZATION OF MUSCA AUTUMNALIS DEGEER BY THELAZIA SP. IN MASSACHUSETTS WITH OBSERVATIONS ON MUSCA DOMESTICA L. AS AN UNSUITABLE INTER¬ MEDIATE HOST. 29
Introduction. 29 Materials and Methods ...... , , 30 Results .. 38 Discussion and Conclusions ...... 50
III. BOVINE THELAZIASIS IN MASSACHUSETTS: FIRST REPORT IN THE NORTHEASTERN UNITED STATES .. 55
Introduction ...... 53 Materials and Methods ...... 57 Results. 62 Discussion .. 71 Conclusions. 74
IV. DEVELOPMENT OF THE BOVINE EYEWORM, THELAZIA GULOSA (RAILLET AND HENRY), IN EXPERIMENTALLY INFECTED, LABORATORY-REARED, FEMALE MUSCA AUTUMNALIS DEGEER. 76
Introduction. 76 Materials and Methods . 78
vii Results .. 81 Discussion ...... 83 Conclusions.... 90
LITERATURE CITED . . 91
viii LIST OF TABLES
1. Percentage parasitism of female face flies by immature nematodes of the genus Thelazia, and parasite load of infected flies (1976).... 37 2. Percentage parasitism of female face flies by Thelazia sp., parasite load of infected flies and percentage of total and parasitized flies parous (1977).. 38 3. Percentage parasitism of female face flies by Thelazia sp., parasite load of infected flies and percentage of total sind parasitized flies parous (1978)... 42 4. Predilection sites of capsule attachment by Thelazia sp. in parasitized female face flies collected from cattle in Massachusetts (1976-1978) . . 48 5. Distribution of capsule attachment sites of Thelazia larvae in multiply parasitized female face flies collected from cattle in Massachusetts (1976-1978) .... 49 6. Measurements of adult bovine Thelazia worms from a Massachusetts abattoir survey (1977-1978) .. 67 7. Measurements of key morphological features of Thelazia gulosa in experimentally infected, laboratory-reared, female face flies (Musca autumnalis) 88
ix LIST OF FIGURES
1. Geographic distribution of face fly populations associated with cattle sampled in 1976 survey for parasitization by Thelazia sp. in Massachusetts ...... 32 2. Second instar Thelazia larva in capsule attached to fat body. 3^ 3. Third instar invasive larva in capsule. 3^ 4. Capsule recently evacuated by invasive larva showing site of exit. 3^ 5. Temporal patterns of parasitization of female face flies by Thelazia sp, at three dairy farms in Western Massachusetts during the summer of 1977 . 40 6. Percentage parasitism of female face flies by Thelazia sp. and percentage parous flies collected from Herd C during the summer of 1978 . .... 44 7. Parasite load of field-collected female face flies parasitized by Thelazia sp. in Massachusetts (1976-1978). 47 8. Bovine eye everted through lids to illustrate location of lachrimal ducts, preferred site of Thelazia gulosa .... 59 9. Bovine eye everted through lids to illustrate location of the third eyelid. skrjabinl is most often found in ducts originating at the base of this structure ...... 59 10. T^ gulosa parasite laid across a bovine eye to illustrate size of the nematode relative to the eye ...... 6l 11. Abscess on surface of conjunctiva from which a nodule of T. gulosa worms was removed . . 6l 12. Thelazia gulosa, anterior end of female. 64 13. Thelazia gulosa, posterior end of female. 64 14. Thelazia skrjabini, anterior end of female. 64 15. Thelazia skrjabini, posterior end of female . .. 64 16. Thelazia gulosa, posterior end of male . 66 17. Thelazia skr jabini, posterior end of male . , . . . . 66 18. Parasite load of Thelazia eyeworms observed in bovine eyes from Massachusetts abattoirs .. . 70 19. A portion of the anterior region of a sexually mature female T. gulosa parasite showing ovaries packed with nematode larvae . .. 80 20. A recently hatched T^ gulosa larva with egg shell clinging to posterior end 80 21. Tj_ gulosa larva from face fly at 3 days post- infection .. 85 22. T^ gulosa larva from face fly at 6 days post¬ infection ...... 85 23. Anterior end of gulosa invasive larva from face fly at 9 days post-infection . 87 24. Posterior end of gulosa invasive larva from face fly at 9 days post-infection .. • 87
x CHAPTER I
LITERATURE REVIEW
The Face Fly, Musca autumnalls DeGeer a j l - - - -———
Life history. The face fly, Musca autumnalls, is an imported pest of livestock which was first reported in North America in Middleton, Nova
Scotia in 1952 (MacNay, 1952), where it is thought to have been intro¬ duced ca. 1950 via the luggage of Royal Canadian Air Force men return¬
ing from a tour of duty in Europe (Davis, 1961), Since its introduction
it has dispersed into all but the most arid regions of the continent
north of Mexico.
Female face flies obtain nourishment by feeding on bovine feces and
the various secretions of the body of the host, including lachrimal,
nasal and salivary secretions as well as blood and pus emanating from
wounds and feeding punctures of biting flies (Teskey, i960). Males are
rarely found feeding on the animals and, although little work has been
done on their feeding behavior, it is generally thought that they are
primarily nectar and pollen feeders, occasionally supplementing their
diet with bovine feces (Matthysse, 1962; Miller and Treece, 1968a; Tes¬
key, 1969).
Miller and Treece (1968a) studied patterns of feeding behavior in
female face flies in the laboratory and in the field. In the laboratory,
a cyclic pattern of feeding choices was observed j peak blood feeding was
noted in flies at 3 and 8 days post-emergence while maximum feces feeding
occurred on days 4, 7, 10 and 13. In the field, the highest incidence of
1 2
feeding on nasal and lachrimal secretions correlated with that of blood feeding In the laboratory, on days 3 and 8, Feces feeding was greatest 1 day before and 2 days after peak oviposit ion (Miller and Treece, 1968a).
Mating occurs 3-7 days post-emergence, with females mating but once for life and males attempting to mate frequently (Teskey, 1969). Males perch on conspicuous objects in the field such as fence posts, rocks and isolated shrubs and fly towards any moving object approximating the size of a female fly to attempt copulation (Teskey, i960, 1969). Tobin and
Stoffolano (1973) have studied the courtship of face fly, and a female sex pheromone has been discovered (Chaudhury et al., 1972) and identi¬ fied (Uebel et ad., 1975).
When ovarian development is completed, female flies seek out bovine feces for oviposit ion. The first egg batch is laid on day 6-7, the sec¬ ond and third on days 10-11 and 13, respectively (Miller and Treece,
1968b).
Flies oviposit only into fresh feces. In Denmark, Hammer (in Teskey,
1969) observed oviposit ion in manure up to 24 hours old; however, Teskey
(1969) found that on hot sunny days the manure surface crusted over to an
extent sufficient to prohibit oviposition in 1 hour, while on cloudy,
humid days manure was acceptable for up to 3 hours. Treece (1966) and
Ruprah and Treece (1968) found bovine diet to have an influence on the
attractiveness of manure to female face flies, with animals fed on al¬
falfa and grass producing more attractive feces than animals fed on com
silagej these factors had no effect on larval survival. While the indi¬
vidual factors involved in feces attractiveness have yet to be determin- 3
ed, Bay and Pitts (1976) have shown that "both olfaction and contact are
essential to oviposition stimulation, and have located the presence of the receptors involved in feces recognition on the antennae.
Eggs are deposited 1 to 3 at a time in batches of about 20, and
hatch within 24 hours of deposition (Wang, 1964> Smith et al., 1966).
The number of eggs laid per female is apparently not influenced by the
number of eggs already present on an individual dropping (Papp, 1976).
At optimum temperatures of 35-40°C larval development requires 2 l/2
days (Wang, 1964), Moisture content of the manure is critical to larval
survival. Bay et al. (1969) reported that a fecal moisture content of
ca. 85$ promoted optimum survival and development; moisture contents of
65 and 95$ were lethal. The sex of the host has been found to have no
effect on larval mortality (3ay et al., 1970).
When larval development is completed, the third instar larvae leave
the feces to find a suitable pupation site, generally in the top l/4 inch
of the soil within 4 feet of the dropping (Teskey, 1969). Where the soil
is unacceptable for pupation, mature larvae have been observed to travel
as far as 30 feet in search of a more favorable location (Jones, 1969).
In the field the pupal stage requires 7.5-8 days and in the labora¬
tory, under optimal conditions of 35-40°C and 50—7Q$RH, 4,5-5.5 days
(Wang, 1964). Adults emerge in the morning hours and imbibe water for
the first 2 days post-emergence (Wang, 1964), and then commence the cyc¬
lic feeding pattern cited above (Miller and Treece, 1968a). Thus the de¬
velopmental time from egg to adult is ca. 10-14 days (Wang, 1964; Smith
et al., 1966). 4
Face fly has a facultative reproductive adult diapause which is in¬ duced by a combination of short photoperiod and low temperature (Stoffo- lano and Matthysse, 1967). Newly emerged adults (less than 2 days old) are susceptible to diapause induction and must be exposed to inductive conditions for 20 days for full diapause to occur (Valder et al., 1969).
With the onset of winter, unmated, diapausing flies seek refuge in vari¬ ous structures such as bams and attics, where on warm days they may sun themselves or make short flights outdoors (Smith et al., 1966; Teskey, i960, 1969). In spring, diapause is broken, eggs axe matured and mating takes place j Teskey (1969) observed face flies feeding as early as 5
April in Guelph, Ontario.
Economic importance. Despite the place generally accorded face fly as a major pest of cattle, little accurate information is available on econo¬ mic injury sustained in infested herds. The USDA estimated in 19^5 that
68 million dollars were lost annually to face fly pressure due to de¬ creased meat and milk production (USDA, 1965)* presumably as a result of diversion of grazing time to fly evasion tactics. Treece (1966) noted cattle huddling together to attenuate fly pressure, a phenomenon also noted in wild American bison in Montana and Wyoming (Burger and Ander¬
son, 1970). Schmidtmann (1978) conducted an ingenious experiment to as¬
sess the impact of face fly populations on grazing time by implanting
electronic recorders on the muzzles of cattle in two herds in New York
state, one subject to high and one to low fly pressure. While the herds
showed marked differences in the number of grazing intervals per day, no
significant difference was observed in the total amount of daily grazing 5
time or in the average daily weight gain over a 2 l/2 month period
(Schmidtmann, 1978).
Face fly perhaps has its most deleterious effects on cattle via the transmission of, or predisposing animals to, invasion by pathogenic or¬ ganisms, Jov^iev and Lav^iev (1972) were able to demonstrate transmis¬ sion of a pathogenic strain of coli (no. 078) by feeding adult flies on the feces of infected animals and allowing the flies access to unin- V ' **... fected calves, Steve and Lilly (1965) presented compelling evidence for the ability of face fly to transmit Moraxella bovis (Hauduroy), one of the major causative agents of infectious bovine keratoconjunctivitis
(KBKC), or pinkeye, Cheng (1967) and Brown and Adkins (1972) found strong correlations between face fly populations and IBKC while Burger and An¬ derson (1970) found a similar association with eye disorders of American bison. Shugart (1978) exposed enclosed cattle to face flies infected with
M, bovis; 6l$ of the animals developed clinical IBKC.
As part of the same study Shugart (1978) released face flies infec¬ ted with infectious bovine rhinotracheitis (IBR) virus into a group of enclosed cattle and produced systemic respiratory infections in 57% of the animals. Included in the clinical course of IBR infections is an ocular stage with symptoms virtually identical to those of IBKC (Wil¬ cox, 1968). Pinkeye-like pathology due to IBR frequently occurs as the only clinical sign of infection where there is no respiratory involve¬ ment (Cuthbertson and Wood, 1979).
Shugart (1978) also assessed the impact of aseptic flies on the eyes of cattle. When subjected to an average fly pressure of one face fly/eye/day for 33 days, 75% of the animals* eyes developed petichial 6
lesions, of which 38% progressed to the ecchymotic stage, at which point the eye is more susceptible to invasion by pathogenic organisms.
The economic losses sustained in herds with pinkeye is substantial.
Frisch (1975) found that Hereford Shorthorn cattle with I3KC were 22.8kg
lighter than uninfected animals after 15 months, Peterson and Meyers
(1978) estimated a 10-15% weight reduction in calves infected with IBKC.
Finally, face fly has been demonstrated to vector bovine eyeworms
of the nematode genus Thelazia (Vilagiova, 1962, 1967, 1968). It is this
aspect of the fly's biology which forms the raison d'etre of this thesis.
The Genus Thelazia Bose
Systematics. As part of a larger revision of the superfamily Thelazioi*
dea, Skrjabin et al. (1967) have recently revised the genus Thelazia,
removing a number of avian parasites which were elevated to separate
generic status (Thelaziella) and leaving only the mammalian parasites.
The authors proposed the following, currently accepted, classification
scheme:
Phylum Nematoda
Glass Phasmidea
Order Spirurata
Superfamily Thelazioidea Sobolev
Family Grassicaudidae Skrjabin and Andreeva
Desmidoc ereidae Gram
Gonglyonematidae Sobolev
Pneumospiruridae Wu and Hu
Rhabdochonidae Skrjabin Rictulariidae Ralllet
Spinitectidae Skrjabin et al
Thelaziidae Skrjabin
Subfamily Oxyspirurinae Yamaguti
Thelaziinae Baylis and Daubney
Genus Ceratospirura Schneider
Hempelia Vaz
Thelazlella Skrjabin et al
Thelazia Bose
Description. Nematodes of medium size. Buccal opening or¬ bicular or hexagonal. Four pairs of papillae of external and three of internal circlets. Buccal capsule rectangular, walls thick and edentate. Tail rounded at end, short. Body covered by cuticle with annulations, some¬ times inconspicuous. Annulation continues at anterior part of body, subsequently interrup¬ ted by lateral fields usually starting in the region immediately behind the nerve ring. Eso¬ phagus not divided into muscular and glandular sections. Male, Spicules usually vary greatly in length and structure* the left slender and long, the right short and massive. Tail with ventral involutions, numerous pairs of ventro¬ lateral papillae and also unpaired papilla lo¬ cated anterior to cloaca. Gubemaculum absent. No caudal alae. Female. Vulva in esophageal region. Vagi¬ na long, muscular. Uterus opisthodelphic. Eggs contain embryo, shells thin. Parasites of conjunctival cavity and lach- rimal gland ducts of mammals. Type species* Thelazia rhodesii (Desmarest)
- Skrjabin et al,, 1967, p. 12 8
Definitive hosts.
* Parasite Species Hosts Source
T. brevispiculata Yang and Wei cattle Skrjabin et al., 1967
T. calif omiensis Price man, dog, cat, rabbit, Weinmann et al., 1974 fox, raccoon, deer, bear, coyote, sheep
T. callipaeda Raillet and man, dog, cat, rabbit, Skrjabin et al., 1967 Henry fox, raccoon
T. ershowi Oserskaja swine Skrjabin et al., 1967
T. ferrulata Yang and Wei cattle w
T, gulosa (Raillet and Henry) cattle, yak It
T, hsui Yang and Wei cattle It
T. iheringi Travassos agouti It
T. janikurgani Satubaldin sheep Satubaldin, 1973*
T. kansuensis Yang and Wei cattle Skrjabin et al., 1967
T. lacrymalis (Gurlt) horse, donkey H
T. leesi Raillet and Henry camel •i
T. petrowi Tuchmanjanz and cattle ti Schachurina
T. rhodesii (Desmarest) cattle, zebu, buffalo, ii bison, horse, goat
T. skrjabinl Erschov cattle "
Thelaziasis in North America
T. califomiensis. The first report of Thelazia in North America was made
by Allerton (1929)# who recovered eyeworms from a domestic dog outside
Los Angeles, California. Initially these parasites were identified as the
oriental eyeworm, callipaeda, however Price (1930, 1931)» after exami- 9 nation of additional material from dogs, determined that the nematodes formed a new species and described them as califomiensis. Subsequen¬ tly, canine thelaziasis was reported frequently, with 9 definite cases having been reported by 1937 (Kofoid et al., 1937).
Kofoid and Williams (1935) made the first report of cal if omi¬
ensis in a human; as of 1975 ten cases of human thelaziasis due to this
parasite had found their way into the literature (Knierim and Jack,
1975). This figure is certainly low and probably only poorly reflects
the actual number of cases treated. Lee and Parmelee (1958) found that
by corresponding with three physician colleagues in California they were
able to unearth three previously unreported cases of human thelaziasis,
and stated that "undoubtedly cases of human infections by Thelazia are
more common than might be suspected from the paucity of reports in the
literature" (Lee and Parmelee, 1958).
Virtually all of the reported instances of human infections in the
U.S, were preceded by the patients* having encountered a small dipteran
while visiting or residing in mountainous, rural areas of the states of
the Western seaboard (Kofoid and Williams, 1935» Hosford et al., 1942;
Lee and Parmelee, 1958; Knierim and Jack, 1975)» suggesting that neither
man nor the domestic dog is the principal natural host of califomi-
ensls. To date, the parasite has been recovered from cats, sheep, horses,
black bears, foxes, coyotes, raccoons, rabbits and deer and has been
found in California, Oregon, Nevada and New Mexico (Knapp et al., 1961;
Weinmann et al,, 197*0.
Beitel et al. (1974) conducted a survey of T. califomiensis in 10 black-tailed deer at three locations in Northwestern Oregon. These work¬ ers reported an average infection rate of 32.8$ of the animals surveyed and, in contrast with human and canine infections, found all infections to be asymptomatic (Beitel et al,, 197*0.
The vector of T^ calif ornlensls eluded workers on the west coast for many years. Burnett et al. (1957) found developing parasite larvae in experimentally infected, laboratory-reared Fannia canicularis Malloch, and in benjamini Malloch collected from an endemic zone of the para¬ site in California. Circumstantial evidence also led Winkler and Wagner
(1961) to postulate F^ benjamin! to be the vector, Weinmann et al. (197*+) were unable to observe normal development of the nematodes in experimen¬ tally infected canicularis or in any but a very small number of F, benjamini. Careful observation of the colonies and reexamination of field-collected material revealed that a previously undescribed species of Fannia, more elusive in the field than benjamini and a contaminant of colonies of this species, was the vector (Weinmann et al., 197*+).
Turner (1976) has since described the species as Fannia thelaziae sp.n..
Equine and bovine thelaziasis. In 1966, a horse in Beltsville, Maryland was found infected with specimens of Thelazla not referable to T^ cali- fornlensls, and which were later identified as lacrymalis (Walker and
Becklund, 1971). Barker (1970) found 1\ lacrymalis in a horse in Guelph,
Ontario in January, 1970. Grant et al. (1973) recovered Thelazia sp. from a Washington equine, however, from the description and photograph presented it seems likely that these parasites were T. californlensls.
Lyons and Drudge (1975b) conducted an eyeworm survey of dead horses 11 passing through the diagnostic laboratory of the University of Kentucky and found a surprising 26.*$ of the animals examined to be infected. In a follow-up survey, Lyons et al. (19?6) found 30.3^ of the horses infec¬ ted with T_j_ lacrymalis and demonstrated face fly to be the vector. Vete¬ rinarians in St. Hyacinthe, Quebec, found lacrymalis in the eyes of
horses brought in for cataract operations (Frechette et al., 1976). In addition to these reports equine thelaziasis is said to be present in
Pennsylvania, Kansas and Minnesota (Gelatt, 1973).
Walker and Becklund (1971) recovered specimens of the bovine eye-
worm gulosa from a giraffe at the Los Angeles Zoo which had recently
arrived from Namibia and had been quarantined both in Kenya and Clifton,
N. J. prior to its shipment to the zoo. Chitwood and Stoffolano (1971)
found Thelazia sp. in a female face fly collected from cattle in Amherst,
Massachusetts. A survey of fane flies in two counties in Western Massa¬ chusetts revealed the presence of eyeworm immatures in ,95% of the 2,363 flies examined, however, the species of the nematodes remained uncertain
(Branch and Stoffolano, 197*0.
Lyons et al. (1975) recovered three specimens of Thelazia sp. from
a dead Hereford at the diagnostic laboratory in Lexington, Ky.. In a sub¬
sequent survey of cattle passing through a processing plant in George¬
town, Ky., Lyons and Drudge (1975a) found gulosa and skrjabinl in
15% of the 1?6 animals examined. Since that time, bovine eyeworms have
been found in Quebec (Frechette et al., 1976), Ontario (Surgeoner, pers.
comm.), New York (Schmidtmann, pers. comm.), Wisconsin (Stoffolano, pers,
comm.) and Tennessee (Patton, pers. comm.). 12
V ector-Parasite Int eractions
Intermediate hosts of Thelazia eyeworms. Stoffolano (1970) reviewed the literature on the nematodes associated with the genus Musca. In the table below the known and supposed vectors of Thelazia eyeworms are pre¬ sented, with the information drawn chiefly from Stoffolano*s review ar¬ ticle. More recent information and non-Musca vectors have been added.
Fly Species Parasite Species Source
Musca arnica Zimin Thelazia gulosa Stoffolano, 1970 T. rhodesii it H T, skrjabini ti it Thelazia sp. •i tt
M. autumnalis DeGeer T. gulosa Stoffolano, 1970 T. lacrymalis Lyons et al 1976 T. rhodesii Stoffolano, 1970 T. skrjabini it «•
M. bezzi Patton and Gragg Thelazia sp. Miyamoto et al., 1973
M. convexifrons Thompson T. gulosa Stoffolano, 1970 T. rhodesii it 11 T. skrjabini ti If Thelazia sp. it 11
M. domestica Linnaeus T. gulosa Gupta, 1970 T. rhodesii Stoffolano, 1970
M. hervei Villeneuve T. rhodesii Stoffolano, 1970 T. skrjabini 11 11
M. larvipara Portchinsky T. gulosa Stoffolano, 1970 T. rhodesii it 11 Thelazia sp. i« 11
M. lucidula Leow T. leesi Dobrynin, 1974
M. osiris tfeidemann T. lacrymalis Skr jabin et al., 1967
M. sorbens Wiedemann T. rhodesii Gretillat and Tourre^, 1970
M. tempestiva Fallen T. gulosa Stoffolano, 1970 13
M. vlcina Macquart Thelazia sp. Stoffolano, 1970
M. vitripennis Meigen T, gulosa Stoffolano, 1970 T, skrjabinl M «t
Amiota variegata Fallen T, callipaeda Nagata, i960
A, magna Okada T. callipaeda Nagata, i960
Fannla thelazlae sp.n. T, califomiensls Weinmann et al., 197^1 Turner, 1976
Vector competence. The factors governing the susceptibility of an indi¬ vidual Musca species to infection by Thelazia are not known. Some dispa¬ rity with regard to vector competence appears to occur along geographic lines. Thus the house fly, domestica, is a reported intermediate host of T^ gulosa in India (Gupta, 1970) and of gulosa and T^ rhodesll in
Czechoslovakia (Vilagiova, 1968), while this fly appears to be incapable of supporting eyeworm development in Japan (Miyamoto et al,, 1965)» the
Soviet Union (Klesov, 19^9b) and the U.S, (this thesis). In Japan, M, bezzi has been found to vector eyeworms in some areas (Miyamoto et al,,
1973), but apparently not in others (Miyamoto et al,, 1975). T^ rhodesll is transmitted in the Ukraine primarily by larvipara (Klesov, 1950) and in the Soviet Far East by Mj_ convexifrons (Skrjabin et al,, 1967), although both flies are present in each of these areas.
Perhaps more intriguing are those situations where a number of po¬ tential vector species coexist within an area yet only one or two are involved in eyeworm transmission. Such situations exist in Czechoslova¬ kia (Vilagiova, 1962, 1968) where 2 out of 5 Musca species associated with cattle vector the parasites, and in Japan, where both the number of potential and actual vector species varies considerably from one area to 14 another (Miyamoto, 1965? Miyamoto et al., 1965, 1967, 1975). Similarly, in California, efforts to determine the vector of T^_ califomiensls met with little success until it was determined that a new species of Fannia, and not F^ ben jam ini, F. canicularis or F^ consplcua, was involved in transmission, even though all are found in association with infected ani¬ mals in the field (Weinmann et al., 1974).
A similar situation occurs with other nematode-Musca systems. Pat- naik and Kumar (1972) sampled 10 Musca species observed feeding on ear- sore lesions of buffalo infected with Stephanofllaria zaheeri Singh in
India. Of those 10, only 1 species, autumnalls, was found capable of supporting nematode development. This finding surprised the authors, since in an earlier study in that country only M^ planiceps Wiedemann
had been found to vector the parasites (in Patnaik and Kumar, 1972).
S, assamensis Pande is vectored by Lyperosla titIlians (3ezzi) in the
U.S.S.R. (Ivashkin et al., 1963) and by conducens Walker in India
(Patnaik, 1973). The latter species also transmits S^ kaeli Buckley in
Malaysia (Fadzil, 1975). In the U.S. Stephanofilaria stllesi Chitwood is vectored by the horn fly, Haematobla irritans (L.) (Hibler, 1966).
In South Africa, Nevill (1975) found 3 out of 10 Musca species as¬
sociated with cattle infected with the filarial worm Parafilaria bovi-
cola Tubangui to support development of the parasites to the third in¬
star, Nevill points to the fact that the three species have a close phy¬
logenetic affinity, implying that some genetic factor may predispose
these flies to susceptibility to P^ bovicola. On the other hand, mul-
tipapillosa Pande, a closely related parasite of horses, undergoes de- 15
velopment in Haematobia atripalpls (Gnedina and Osipov, i960).
Because of their greater medical and veterinary importance, a large body of information exists on the interactions of the filarial worms Wu- chereria bancrofti (Cobbold), Brugia sp., and Dirofilaria lmmltla (Lei- dy), with their culicid hosts. Vector competence in some of these sys¬ tems has been demonstrated to be under genetic control, with consider¬ able variability between populations of the same species. McGreevy et al. (197*01 testing the susceptibility of 9 strains of Aedes aegypti
(L.) to parasitization by immitis, found 3 completely refractory strains, 5 of intermediate susceptibility and 2 strains that were 86-89% susceptible. Similarly, experimental infections of different strains of
Ae, aegypti (Theobold) with bancrofti showed refractory and suscep¬ tible strains of each species to the parasite (Partono et al., 1977).
Susceptibility within a vector species has been demonstrated to be governed by a single, recessive, sex-linked gene for Culex pipiens L. to
Brugia pahangi (Buckley and Edeson) (Obiamwe and MacDonald, 1973). A si¬ milar phenomenon has been shown with Ae. aegypti and Dirofilaria immitis
(McGreevy et al., 197*0. Partono (1979) has presented evidence that the genetic mechanism regulating susceptibility of Culex pipiens fatigans
Weidemann to Wuchereria bancrofti is multifactorial.
McGreevy et al. (197*+) emphasized the fact that the vector-parasite relationship is dynamic, and that the vector status of a given species is the result of the sum effects of the susceptibility of the arthropod and the infectivity of the parasite. In some instances the latter factor may be more crucial than the former. Culex pipiens qulnquef asciatus Say 16
is able to vector an urban strain of bancrofti but is refractory to rural strains (Wharton, 196l). Anopheles campestrus Reid is susceptible to periodic but refractory to subperiodic strains of Brugia malayi (Brug)
(Reid et al., 1962), Lawrence and Pester (1967), in a series of experi¬ mental infections spanning 4 generations of the vector, concluded that the increasing susceptibility of Aedes togoi to Brugia patei over time was due to selection pressures on the parasite, rather than the genetics of the vector.
Site of development in the intermediate host. So little is known about the interactions between eyeworms and their intermediate hosts that agreement is lacking even on the mode of parasite development in the fly. The Soviet worker Krastin (1949)> who was perhaps the first per¬ son to study this aspect of the life cycle of eyeworms, referred to T. rhodesii larvae developing in connective tissue capsules in the abdomen of convexifrons, however, no details were given. In subsequent years, many reports pointed to nematode development within the egg follicles of female flies. Krastin (1950) found Thelazia sp. in the egg follicles of
M. arnica, while Klesov (1950) reported rhodesii to develop in the same manner in larvlpara,Krastin (in Skrjabin et al., 1967) found develop¬ ment of rhodesii to occur in the egg follicles of female convexi¬ frons, an observation also noted with gulosa in this fly (Skrjabin et al., 1967). Other Soviets found skrjabini to develop in similar fash¬ ion in arnica and vitripennis (Skrjabin et al., 1967), Nagata (i960) found callipaeda larvae developing in the ovaries of female, and tes¬ tes of male, Amlota variegata and Aj_ magna in Japan. Kozlov (in Skrjabin 17
et al., 1967) reported, in the U.S.S.R., that the same parasite developed in the testes of male Phortica (Amlota) variegata, but that in females development occurred in capsules formed by, and attached to, fat body. On the other hand, Vilagiova (1962, 1967) found that Tj_ gulosa, T, skrjabinl and T_j_ rhodesii develop in the egg follicles of female, and in fat body of male, autumnalis in Czechoslovakia.
Curiously, in the intervening years since 1967 no further reports of eyeworm development in reproductive tissues have been made. The Sovi¬ et worker Dobrynin (197^) 1 in an experimental study with lucidula and
T. leesi, the dromedary parasite, found all capsules attached to fat body. Shinonaga et al. (197^)» in Japan, reported Tj_ rhodesii and T. skrjabinl to develop in capsules attached to the abdominal body wall, fat body and malphigian tubules of hervei. Miyamoto et al, (1973)» also in Japan, found all Thelazla capsules adhering to the abdominal body wall of hervei and bezzi.
Stoffolano (1970) suggested that capsule formation arises an the result of a modified host reaction on the part of the vector, however the origin, structure and function of these capsules remain a mystery despite the emphasis placed on future research in this area by workers in the eyeworm field (Stoffolano, 1970; Miyamoto et al., 1973? Shinona- ga et al., 197*0.
Time of development to the invasive stage larva. Vilagiova (1967) found that development of T. gulosa to the third instar required 28-32 days in
M. autumnalis, and that skrjabinl never reached this stage during that time. Development of T, rhodesii to the invasive stage larva reportedly 18 takes 15-30 days in Mj. larvdpara (Klesov, 1950), ca. 30 days in con- vexlfrons (Skrjabin et al. 1967), 28-32 days in M^ autumnalis (Vilagio- va, 1967), and up to 50 days in hervei (in Shinonaga et al,, 1974).
Dobrynin (1974) found the molt to the third instar of leesi in lu- cidula to occur on day 10-11 at 21-32°C, while Kozlov (in Skr jabin et al., 1967) found callipaeda to require 19-22 days in Phortica (Ami- ota) varlegata, Lyons et al. (1976) observed lacrymalis invasive stage larvae in the head of autumnalis on day 12-15, while califomlensis requires ca. 30 days for such development in Fannla thelaziae (Weinmann
et al., 1974* Turner, 1976). The lack of information on environmental conditions during virtually all of the above studies renders meaningful comparison impossible.
Nevill (1975) conducted experimental infections of Parafilaria bo- vicola in three Musca species suspected of vectoring this parasite in
South Africa. At 27°G and 50-70^RH, P^ bovicola developed to the inva¬
sive stage in 14 days in lusoria Wiedemann, xanthomeles Wiedemann and Musca sp.n. (Nevill, 1975). Stephanofilarla stilesi invasives were
found in the head of Haematobia irritans at 18-21 days when maintained at 27°G and 40-8Q$RH (Hibler, 1966). Invasive larvae of S^ assamensis were found in the head of Musca conducens at 20 days (Patnaik and Roy,
1966) 1 the molt to the third instar occurs on day 13-15 at 25°C (Pat¬ naik, 1973). S_j_ kaeli develops to the third instar in 8 days and is
found in the head of conducens at day 10 (Fadzil, 1975).
Steward reported that Onchocerca gutterosa larvae were in the head
of Simulium omatum Meigen at 19 days post-infectionj the second molt
had occurred on day 15 (Steward, 1937). More recently, Mellor (1975) in- 19
feeted Cullcoides nebeculosis Meigen and variipennls Meigen with 0.
cervicalis Raillet and Henry via a membrane feeding technique. When
maintained at 23^0 and 80$RH, invasive larvae were found in the heads
of infected flies by day 14-15 (Mellor, 1975),
In susceptible strains of Aedes aegypti maintained at 2?°G and 70-
9<$RHf McGreevy et al. (1974) found Dirofllaria immitis to develop to
the third instar in 10-13 days,
Wuchereria bancrofti reaches the invasive stage larva in 13 days in
Anopheles funestus Giles (in Krasfur and Garret-Jones, 1977) and, at 27-
32°C and 75$RH, Brugia malayi and pahangi reach the invasive stage in
9 days in susceptible strains of Aedes aegypti (Beckett and MacDonald,
1971).
Pathology, Diagnosis and Treatment of 3ovine Thelaziasis
Pathology.
General considerations and mechanisms. Pathology associated with bovine thelaziasis is highly variable, ranging from sub-clinical (Lyons and Drudge, 1975*>j Arbuckle and Khalil, 1976, 1973) to sufficiently se¬ vere as to result in 10-15$ mortality in calves (Gretillat and Tourre",
1970). Where present, clinical symptoms of eyeworm infections may in¬ clude profuse lacrimation, photophobia, epiphora, conjunctivitis, muco¬ purulent discharge, corneal ulceration and opacity, and blindness (Stof- folano, 1970). Histopathological examination of lachrimal ducts contain¬ ing eyeworms by Chauhan and Pande (1973) revealed desquamation of the superficial tissue layers, hypertrophy of the lymphoid follicles, con¬ gestion of surrounding blood vessels, and damage and displacement of the 20 soft areolar connective tissue.
The mechanism of pathogenicity of Thelazia infections is not known.
Workers in the Soviet Union have attributed most of the symptomology to the invasive larva, suggesting that either metabolic substances produced by the parasites or mechanical irritation due to the strong cuticular striations of the nematode result in the disorder (Krastin and Ivashkin,
19495 Klesov, 1949af Ivashkin, 1953). The larvae are present in the eyes
of the host in greatest numbers in mid-summer, and Arbuckle and Khalil
(1976) implied that this might have partially explained why little pa¬
thology wan observed in a winter abattoir survey in Britain, while ear¬
lier reports in that country during summer months had noted many of the
classical symptoms of thelaziasis (Mitchell and Hughes, 1953I Fitzsimmons,
19635 Oakly, 1969). This hypothesis, however, could not be confirmed in
a later survey which spanned an entire year (Arbuckle and Khalil, 1978).
Another proposed cause of pathogenicity is that the parasites may
create conditions in the tissues of the eye which predispose them to
secondary invasion by the etiological agents of I3KC (Fitzsimmons,
I963). Aruo (1974) made eye-swabs of Ugandan cattle afflicted with T,
rhodesii and found Proteus vulgaris and coll, both of which have been
isolated in cases of IBKC (Wilcox, 1968). In a study of IBKC in cattle
in Rostock, Prange et al. (1968) found a significant correlation between
Thelazia sp. and alphahemolytic diplococci in the eyes of symptomatic
animals. Treatment of infected eyes with antibiotics, however, had no
effect on the condition, implying that the parasites may play a larger
role in the development of symptoms. Indeed, Paul and Dulceanu (1971),
in Rumania, were able to demonstrate histologically that the nematodes 21
were directly responsible for the appearance and development of lesions.
Efforts to determine the true pathological significance of eyeworms have been hampered by the overlap of symptomology and time of infection of thelaziasis with IBKG (Stoffolano, 1970), a condition obscured by the numerous causative agents involved. Morgan (1977) has emphasized the need for a careful evaluation of the many proven and supposed etiological agents of IBKG, which include Koraxella bovis, Lysteria monocytogenes,
Mycoplasma sp., M_j_ bovlrhinis, M. laidawli, Rickettsia-like organisms, psittacosis-lymphogranuloma-trachoma organisms, viruses, and abiotic
factors (Wilcox, 19685 Bedford, 1976). Separation and analysis of these various factors will certainly prove a most formidable task. For exam¬
ple, M^ bovis, generally regarded as the single most important causa¬
tive agent of I3KC (Hughes and Pugh, 1971* 1972; Morgan, 1977)» is a com¬
mon bacterium of the conjunctival sac of completely asymptomatic animals,
and it has been suggested that this organism may act principally as a
synergist with other factors (Bedford, 1976).
In addition to the veterinary importance of eyeworms, Ivashkin
(1953) stresses the economic injury sustained in infected herds, even
where pathology is rather mild, stating that "lactating dairy cows ...
stop lactation almost completely and lose condition to a marked degree
in the diseased state”. Similarly, Amo (197*0 found that animals treat¬
ed for rhodesil infections increase their grazing time substantially,
and Gunyavyi (1969) reports that the cost of prophylactic treatments for
thelaziasis is one fiftieth the amount lost in untreated herds due to
decreased meat and milk production. It has been shown with other hel¬
minth parasites of dairy cattle that subclinical infections may result 22
in substantial economic loss (Gutierres et al., 1979),
Variability and its causes. Of the three most prevalent and, hence,
most studied, eyeworm species, gulosa, T, skrjabini and rhodesii,
the latter is consistently the most pathogenic, rhodesii infections
result in 10-15/6 mortality among calves in Senegal (Gretillat and Tourre^
1970) and, in addition to generally producing the entire spectrum of symp¬
toms of thelaziasis, frequently terminate in corneal ulcerations and per¬
manent blindness (Divljanovic, 1958} Vorhadsky, 1970), Niimi (in Okoshi
and Kitano, 1966a) reported a morbidity rate of 10^ of infected bovines
in Japan, Although more recent studies in that country would indicate that this figure is somewhat inflated, Japanese cattle infected with 1\ rhodesii display lacrimation, conjunctivitis and photophobia (Okoshi and
Kitano 1967) while parasitism by gulosa and skrjabini is predomi¬ nantly sub-clinical (Miyamoto et al., 1974). Cock (1972) reported squa¬ mous cell carcinoma associated with rhodesii infection in Rhodesia,
Pathogenicity arising from Tj_ gulosa and skrjabini infections, although more variable, is generally less severe than that of T. rhode¬ sii, Working in Mongolia, Ivashkin (1953) described the clinical course of infection by these two parasites. The initial symptons, in July, were characterized by excessive lacrimation and conjunctivitis, followed by progressive stages of keratitis. 3y late August, the inflammation gradu¬ ally declined, with most animals recovering normally in subsequent weeks
(Ivashkin, 1953). Chauhan et al. (1976) found skrjabini parasites in the vitreous humour of an infected animal. Gorodovich (1963) reported, in a therapeutic evaluation experiment in Amor province, U.S.S.R., that during June-August 79. of untreated cattle infected with gulosa and 23
T. skrjablni were symptomatic. In Israel, Franzos (1964) found mucopuru¬ lent discharge, conjunctivitis, and abscess formation on the conjunctiva and eyelids in cattle with gulosa infections. Hovorka and Corba (1970) found T_j_ gulosa infections associated with keratoconjunctivitis in calves in Czechoslovakia. Nonetheless, the corneal ulcerations and permanent blindness reported with rhodesii axe rarely, if ever, present in T. gulosa or skrjabini infections. Indeed, symptomology associated with such infections is mild in Germany (Eckert et al., 1964), and almost en¬ tirely sub-clinical in the U.S. (Lyons and Drudge, 1975a), Britain (Ar-
« buckle and Khalil, 1976, 1978; Turfey and Chandler, 1978), and Japan
(Miyamoto et al., 1974).
As indicated above, a substantial degree of pathogenic variability occurs along geographic lines. The reasons for such variability are not known. Hovorka (1959) found both the incidence and intensity of rho¬ desii infections in Czechoslovakia to vary with the landscape, being highest in wooded lowland regions and lowest in the highlands. This finding led him to postulate that air temperature and moisture were cri¬ tical factors. Vilagiova (1968) confirmed this variability, however, she demonstrated that this was due more to differences in the size and spe¬ cies composition of the Musca populations from one region to another, rather than to any direct effects of climate on pathology.
A generally accepted tenet of host-symbiont interactions is that the extent of pathogenicity is related to the evolutionary age of the association, with more severe symptomology suggestive of a fairly rece¬ ntly established relationship (Schmidt and Roberts, 1977). A small body of evidence seems to point to the possibility that some of the variabi- 24
lity of thelaziasis-associated pathology may be due to this factor. Iva¬
shkin (1953) noted that T\_ gulosa and skr jabini produced far graver
symptoms in Mongolian yaks, which encounter the parasites only under con¬
ditions of domestication in low-lying areas, than in domestic bovines.
Thelazia califomiensis, the only member of the genus indigenous to North
America, produces no visible effects in populations of wild deer (Beitel
et al., 1974) but causes considerable discomfort to man (Lee and Parme-
lee, 1958* Knierim and Jack, 1975) and domestic dogs (Kofoid et al.,
1937). Tjl rhodesil has been reported from a number of wild game animals
(Skrjabin et al,, 1967; Bindemagel, 1972; Chauhan and Pande, 1973)» and
its movement into domestic cattle is almost certainly a relatively re¬ cent event. Vorhadsky (1970) found that domesticated cattle crossed with zebu showed more severe symptomology than pure-bred zebu.
The degree to which any disease affects its victim is determined in large part by the overall fitness of the individual. A parasite may af¬ flict an otherwise healthy animal to a far lesser extent than an animal already combatting an army of pathogenic organisms (Schmidt and Roberts,
1977). This factor may partially explain why T^ rhodesii appears so vi¬ rulent in tropical areas, particularly in Africa, where the animals are laboring under heavier parasite loads than in more temperate climes.
Conversely, the relatively benign effects of ]\ gulosa and T\ skrjabini in areas such as the U.S., Britain, Western Europe and Japan may be at¬ tributable to the generally better sanitary conditions in the developed world, coupled with the overall smaller number of parasite species favo¬ red by these temperate areas. 25
Diagnosis, A final confounding factor in the effort to determine the
true pathogenic importance of thelaziasis is the lack of an effective
ante-mortem diagnostic technique. Because of their hidden location in
the eye, the presence of adults can not be determined on gross examina¬
tion (Turfey and Chandler, 1978). For this reason, detection of eyeworms
in the living animal has been restricted to examining eyewashings for
the presence of first-instar larvae liberated from mated, adult female
eyeworms (Stoffolano, 1970). This technique, however, is effective only at certain times of the year, it does not detect the presence of unmated or immature parasites, and is very laborious. Arbuckle and Khalil (1978) found eyewashings to be completely unsatisfactory in the diagnosis of. thelaziasis in British cattle herds known to include parasitized indivi¬ duals.
Chemotherapy and prophylaxis. Until fairly recently, treatment of Thela- zia infections was restricted to the use of mechanical removal and local antiseptics (Thienpont, 1979). Kofoid et al. (1937) recommended mechani¬ cal removal of callfomiensis and rhodesii followed by irrigation with boric acid solution, while Fitzsimmons (1963) found irrigation of the conjunctival sac with physiological saline to be equally effective.
Knierim and Jack (1975) reported that removal of califomiensis eye- worms from man was sufficient to halt symptoaclogy associated with the parasites. Oakly (1969) also recommended mechanical removal of eyeworms to clear up the immediate pathology, but found that subsequent topical application of the nematocide methyridlne had the additional virtue of preventing reinfection. A Soviet worker in Kazakstan proposed an intri- 26
guing, non-chemical treatment-prophylactic for bovine thelaziasis: Satu- baldin (1973a) reported that cauterization of the inner surface of the nictitating membrane with an electrode at 1000°G both destroyed all pa¬ rasites present in the eye and conferred resistance to future infections for the following 18-20 months.
Fitzsimmons (19&3) has emphasized the importance of correct species identification of the causative agents of thelaziasis prior to selecting a course of treatment. Thus rhodesii and califomiensis, which ty¬ pically axe found under the third eyelid or in the conjunctival sac, may be mechanically removed, while gulosa and I\_ skrjabini pose greater' problems by virtue of their more secretive locations in the lachrimal and nasolachrimal ducts.
Klesov (1949a) reported that irrigation of the eyes 3 times in suc¬ cession with 50-7Occ of .05% iodine/. 0757o potassium iodide was quite ef¬ fective against all eyeworms, a finding supported by Shanzel and Hegero- va (1961). Krastin and Potilina (1956) found irrigation of the eye with
.5% lysol solution was preferable to the recommended iodine treatment.
Rauchbach (1958) pointed out that the effectiveness of iodine solution could be augmented by backflushing of the nasolachrimal duct via a canu- la inserted into the nostril of the animal. Similarly, Hovorka (1951) recommended backflushing, rather than irrigation, with .0% Lugol's so¬ lution,
Kostyra (i960) found that irrigation of the conjunctival sac with a
37° solution of piperazine adipate and J/o boric acid was effective while
Sobolewska and Gajda (1970) recommended irrigation with J7° boric acid
Plus any of the following* irrigation with J/o piperazine adipate; 1% pen- 27 icillin ointment; 1% iodeforin; or 6% calomel ointment.
Meshchaninov and Enilin (1962) reported 3-^1 of 1$ trichlorphon applied to the conjunctival sac in conjunction with topical application of a 1% trichlorphon ointment gave excellent control of eyeworms. Augua- dra (1962) also found trichlorphon to be effective when administered orally at 10-20g per animal for 10 days. In addition, he reported that piperazine succinate added to feed at lOOmg/kg body weight for 3 days gave satisfactory control; intravenous administration of 20-30ml of 10% suramin was completely ineffective (Auguadra, 1962).
Corba (1969a) tested 8 anthelminthics against Thelazia larvae and adults in vitro. The results of that study indicated that trichlorphon, methydrine, and tetramisole were most effective; piperazine adipate, di- thiazanin iodide, diethylcarbamazine citrate, ditrazine phosphate, and cyanacethydrazid were considered unsuitable (Corba, 1969a).
Eastern European and Soviet workers have shown interest in the de¬ velopment of prophylactic as well as therapeutic control strategies.
Gorodovich (1963) found that injection of 5^1 diethylcarbamazine phos¬ phate solution into the muscles around the lachrimal glands for 6 days had both prophylactic and curative properties. Gunyavyi (1969) recommen¬ ded conjunctival administration of 0.5-1.0^ lysol solution in conjunc¬ tion with .25% procaine, followed by removal of dead worms. For both treatment and prophylaxis Arifdzanov et al. (1970) suggested intravenous administration of 1% bigumal solution at 5®g/kg concurrently with 7% diminazene aceturate at 3.5mg/kg.
In the past decade two products, tetramisole and its laevo-rotary isomer, levamisole, have shown great promise in eyeworm therapy. Tetrami- 28
sole, a broad spectrum anthelminthic which has been used against species of Ascaridia, Heterakis, Trichocephalus, Chabertia, Bunostomum, Nemato- spiroides and Trichostrogylus (Bossche and Janssen, 1967), has been de¬ monstrated to be highly effective when administered orally at 15mg/kg
(Corba 1969a,b; Gorba et al., 1969? Hovorka and Gorba, 1970). At this dosage and higher, however, this product results in untoward side effects in the animal, including sharp lowering of the blood pressure and severe hyperesthesia (in Aruo, 197*0 • Aruo (197*+) found that by reducing the dosage to 12.5mg/kg the side effects were lost with no diminution of ef¬ fectiveness,
Levamisole is equally active at much lower doses. Hovorka and Gorba
(1970) and Vassiliades et al. (1975) found that 5®gAg levamisole, taken orally, was as effective as tetramisole at 15mg/kg. Both groups also found that topical application of a levamisole eye lotion gave good control of eyeworms. Michalski (1976) preferred application of 2ml leva- misole directly into the conjunctival sac while Cock (1972) suggested irrigation of the eye with Z.% levamisole hydrochloride diluted 5Q?o in
1% hibitane solution. CHAPTER II
GEOGRAPHIC RANGE AND TEMPORAL PATTERNS OF PARASITIZATION OF
MUSCA AUTUMNAL IS DEGEER 3Y THELAZIA SP. IN MASSACHUSETTS
WITH OBSERVATIONS ON MUSCA DOMESTICA L. AS AN UNSUITABLE
INTERMEDIATE HOST.
Introduction
Members of the dipteran genus Musca have been shown to vector a num¬ ber of mammalian eyeworms belonging to the nematode genus Thelazia (Spi- rurataiThelaziidae). To date, thirteen species of Musca have been demon¬ strated to transmit eyeworms (Stoffolano, 1970; Gretillat and Tourre",
1970; Miyamoto et ad., 1973). The house fly, Musca domestlca L., trans¬ mits the bovine parasites T\_ gulosa (Raillet and Henry) and T_j_ rhodesii
(Desmarest) in Czechoslovakia (Vilagiova, 1964), and has been found para¬ sitized by T_j_ gulosa in India (Gupta, 1970). The face fly, M^ autumnalls
DeGeer, has been demonstrated to vector both gulosa and T. rhodesii in
Czechoslovakia (Vilagiova, 1964), and the equine eyeworm T, lacrymalis
(Gurlt) in the United States (Lyons et al., 1976).
Eyeworm larvae were first found in face flies in North America in
Massachusetts (Chitwood and Stoffolano, 1971) shortly after the first re¬ ported case of equine thelaziasis on the continent (Barker, 1970), A sur¬ vey of fly populations in two counties in Western Massachusetts later confirmed the presence of Thelazia sp. in adult female face flies (Branch and Stoffolano, 1974), however, the species and definitive host of the parasites remained unknown. Surveys of the eyes of slaughtered cattle in
Kentucky (Lyons et al., 1975l Lyons and Drudge, 1975a) and Massachusetts
29 30
(this thesis, Chapter III) have shown the presence of gulosa and T.
skr.jabini in the U. S,, while lacrymalis has been recovered from the
eyes of horses in Kentucky (Lyons and Drudge, 1975b), Maryland (Walker
and Becklund, 1971), Ontario (Barker, 1970) and Quebec (Frechette et al.,
1976).
The objectives of the present study were three-fold? (l) to determime
the range of Thelazia sp. in Massachusetts; (2) to ascertain what tempo¬
ral patterns of parasitization occur in face fly populations associated
with single herds; and (3) to assess the vector potential of house fly.
Materials and Methods
Geographic range. During the summer of 1976 female face flies were samp¬
led from 18 herds of beef and dairy cattle across the state (cf. Fig, l).
Collections were made by presenting cattle with plywood boards freshly
smeared with citrated beef blood and sweeping flies off of the boards
with a net as they alighted to feed. The flies were placed in cages, pro¬ vided with distilled water, powdered milk and granulated sugar and retur¬ ned to a rearing room maintained at 24hr light, 22-27°C, and 40-5Q^RH where they were held 1-5 days before dissection. Prior to dissection,
flies were anesthetized with CO2, immobilized by gently crushing the tho¬ rax and placed in a Syracuse watch glass flooded with physiological sa¬ line (Norman and Duve, 1969). Using forceps, the abdomen was opened along the ventral mid-line and the gut, ovaries and air sacs were removed. Af¬ ter close examination for the presence of encapsulated nematode larvae
(Fig.'s 2-4) the fat body was removed, thus exposing the inner lining of Fig. 1. Geographic distribution of face fly populations associated with cattle sampled in 1976 survey for parasitization by Thelazia sp, in Massachusetts.
Figures 2-4. Examples of Thelazia sp. capsules found in field collected face flies. 2. Second instar parasite larva in capsule attached to fat body. 3» Third instar invasive larva in capsule. 4. Capsule recently evacuated by invasive larva: arrow indicates site of exit, (1 in 53 .196 mm). 34 35
the abdominal wall which was subsequently inspected for capsules. The thorax, head and proboscis were then dissected and examined for the pre¬ sence of 3rd instar, invasive stage nematode larvae. The number of para¬ sites and the location of capsule attachment sites within each fly were recorded. The lack of taxonomic information on immature parasites pre¬ cluded species determinations.
Temporal patterns of parasitization. In 1977 we returned to three of the dairy herds sampled during the previous summer in order to monitor tem¬ poral rates of parasitism in the associated fly populations. The three herds, designated as Herds A, B and C, were all Holsteins located in the western part of the state and correspond to 1976 site numbers 3* 9 and.
18, respectively (Fig. l). Herd A was composed of 20-35 heifers and had shown a high rate of parasitism in 1976. Herd B was a large milking herd of about 300 head with a low incidence of parasitism in the previous year’s survey. Herd G was selected for its small size, 13-16 animals, its moderate rate of parasitism in 1976 and the fact that, unlike Herds A and
B, it was geographically isolated from the next closest face fly popula¬ tion by at least 10 miles of mountainous, wooded terrain, thus eliminat¬
ing the possible confounding factor of the inclusion of flies from nearby
herds in the samples. In addition to the information gathered during the
1976 survey, the ovaries of most flies were examined for parity (Miller
and Treece, 1968b).
Vector potential of house fly. In the summer of 1978 we returned to Herd
G to simultaneously sample both house fly and face fly populations. In
addition to its isolation and small size, this herd was known to be asso- 36 dated with populations of both fly species, each of which made daily contact with the animals. Face flies were sampled in the afternoon while the animals were in a pasture adjacent to the milking bam while house flies were sampled by sweeping the walls, ceiling and floor of the bam.
The flies were brought back to the lab, held 2-5 days in the rearing room and dissected in the manner described above.
Results
Geographic range. Results of the 1976 statewide survey of female face flies for Thelazia sp. are presented in Table 1. Parasitism ranged from
0.4$ at Site 12 on 11 August to 13.2% on the final sampling date of 7
September. Sample sizes ranged from 131 to 532 for an average of 304 flies per sample. A total of 144 parasitized flies were found out of
5,479 flies dissected during the study, yielding an average percentage parasitism of 2.6.
Temporal patterns of parasitization. Results of the 1977 survey of 3 dairy herds for face flies parasitized by Thelazia sp. are shown in
Table 2 and Fig. 5. Herd A had the highest incidence of parasitism
(7.5%). Two peaks of parasitism were observed early in the season, 13.9 and 16,9% on 21 May and 20 June, respectively, followed by a decline to below 5.2$ which persisted through July and August. A late season peak of 10,2$ on 3 September and 11,5% on the final sampling date of 20 Sep¬ tember was observed.
The first sample of flies collected from Herd B on 20 June showed an incidence of parasitism of 7.6$. This was followed by a decline to below 37
Table 1. - Percentage parasitism of female face flies by immature nema¬ todes of the genus Thelazia, and parasite load of infected flies. Collec¬ ted from herds of cattle in Massachusetts (1976).
Collection Date Sample Percentage No. Parasites/infected Fly Site No.a Collected Size ( )b Parasitism Mean (RangeJ
1 7/10 292( 7) 2.4 5.5(3-8 )
2 7/12 15M 1) 0.6 10.0°
3 7/12 300( 3) 1.0 2.3(1-4 )
4 7/16 524( 3) 0.6 4,0(1-9 ) O -3- O 5 7/18 195( 1) 0.5 •
6 7/28 454( 4) 0.9 5.2(1-8 )
7 8/2 397 (2) 0.5 2.0°
8 8/2 200( 4) 2.0 4.8(1-15)
9 8/5 324(17) 5.2 4.4(1-33)
10 8/5 532(14) 2.6 1.8(1-3 )
11 8/11 431(28) 6.5 2.2(1-6 )
12 8/11 26l( 1) 0.4 1.0
13 8/15 213(H) 5.2 2.9(1-8 )
14 8/23 139 ( 2) 1.4 5.0(3-7 )
15 8/23 365(H) 3.0 2.4(1-9 )
16 8/27 423(12) 2.8 2.6(1-8 )
17 8/28 131( 4) 3.0 10.0(2-28)
18 9/7 144(19) 13.2 2.7(1-8 ) Totals 5,479(144) 3.1
a Location of sites presented in Fig. 1. ^ Numbers in parentheses indicate number of parasitized individuals in sample. c One parasitized fly in sample. 38
Table 2. Percentage parasitism of female face flies by Thelazia sp., parasite load of infected flies and percentage of total and parasitized flies parous.a
No. Parasites/ Herd Collection Sample Percentage Infected fly Percentage Parous Flies (1976 Site No.) Cate Size ( ) b Parasitism Mean (Range) Total Parasit. Flies
5/21 187(26) 13.9 4.6(1-18 ) -c _c 6/12 318( 6) 1.9 3.3(1-10 ) -c 66.7 6/20 319(54;) 16.9 3.2(1-11 ) 67.0 92.6 Herd A 7/6 172( 3;) 2.2 2.7(1-5 , ) 44.7 50.0* (No. 3) 7/14- 255(13;) 5.1 1.8(1-5 , ) 47.3 84.6 7/29 256(11;) 4.2 3.0(1-11 ) 27.8 63.6 3/15 298 ( 9 ) 3.0 3.4(1-8 ; ) 37.1 38.9 9/3 305(31, ) 10.2 3.1(1-10,) 30.2 30.6 9/20 148(17; ) 11.5 2.9(1-9 : ) 16.3 7 6.5 Totals 2258(170) 7.5 3.2 337b 7^75
6/l4 604(46) I 7.6 3.8(1-19: ) 46.6 67.4 6/30 293( 9 ' 3.1 3.2(1-9 ; ) 23.7 88.9 Herd 3 7/11 352( 5j ' 3.1 1.4(1-3 ; ) 32.5 30.0 (No. 9) 7/26 279( 0 0.0 33.0 m 8/S 386( 9) 2.3 4.0(1-21) 1 32.4 100.0 3/23 251(13) 5.2 3.2(1-9 ) 1 36.6 69.2 9/10 153( 9) 5.8 3.6(1-9 ) 20.3 66.7 Totals ^28(91) 3.9 3.5 30 7H3
6/6 59( 3) 5.1 3.3(2-6 ) .0 _c 6/20 233(32) 13.7 2.8(1-3 ) 73.2 84.4 7/6, 309(12) 3.9 4.1(1-10) 50.3 91.7 Herd C 7/14 291( 9) 3.1 3.0(1-6 ) 44.6 1C0.0 (No. 18) 8/3 295(12) 4.1 3.3(1-6 ) 50.3 91.7 8/15 237( 9) 3.8 1.8(1-10) 51.5 100.0 9/3 175(11) 6.3 3.3(1-4 ) 38.7 81.3 9/12 181(16) 8.8 3.0(1-9 ) 22.0 75.0 Totals 1780(104 7 ITS 3.0 573 3772
a All flies collected from three dairy herds during the summer of 1977. Numbers in parentheses indicate number of parasitized individuals in'sample ® rarity data not taken. * d One fly also parasitized by Heterotylenchus autumnal is. Fig. 5. Temporal patterns of parasitization of female face flies by Thelazia sp. at three dairy farms in Western Massachusetts during the summer of 1977. 40 Fig. 5 MAY JUNE JULY AUGUST SEPT.
WSUISVHVd 3DVlNBDUBd 41
3.2/6 which was maintained through the remainder of June, July and early
August. On 23 August parasitism increased to 5.2$ and remained high. Of
2,328 flies examined, 91 (3.9%) were parasitized by Thelazia sp. imma-
tures.
On 6 June the incidence of Thelazia infection in the flies associa¬
ted with Herd G was 5.1% and increased to 13.7% on 20 June. A subsequent
decline to below 4,2% was observed which persisted through July and Aug¬ ust, An increase to 6,3% was noted on 3 September followed by a further
increase to 8.8% on 12 September. Of 1,780 face flies sampled from this herd 104 (5.8%) were parasitized.
Vector potential of house fly. During the summer of 19?8, 1,635 female face flies and an equal number of female house flies were sampled from
Herd G. Of that number 93, or 5.7%, of the face flies were parasitized by
Thelazia sp.. Of the house flies none were found with any live para¬ sites, however, two flies contained several melanized, dead nematodes indicative of a host reaction. It could not be determined whether the parasites belonged to the genera Habronema, Heteroty1enchus or Thelazia.
The temporal pattern of parasitization of face fly for 1978 was si- milax to that observed in the previous year. These data are summarized in Table 3 and Fig, 6. High incidences of parasitism were observed early in the summer, with two peaks of 30.2 and 11.3% on 28 June and 7 July, respectively, with a low point of 4.7% in between, on 5 July. For the remainder of July and through August, the percentage parasitism remained below 6.0%. On 9 September an increase to 12.0% was observed. No parasi¬ tized individuals were found in the final sample of 10 October. 42
Table 3. - Percentage parasitism of female face flies by Thelazia sp., parasite load of infected flies and percentage total and parasitized flies parous.a
No. Parasites/ Collection Sample Percentage Infected fly Percentage Parous Flies Date Size ( Parasitism Mean (Range) Total Parasit. Flies
6/21 92(14) 15.2 1.7(1-4 ) 74.4 100.0
6/28 53(16) 30.2 2.8(1-9 ) 73.1 81.2
7/5 107( 5) 4.7 2.6(1-6 ) 33.0 100.0
7/7 106(12) 11.3 1.8(1-4 ) 56.2 83.3
7/13 187( 8) 4.3 5.4(1-15) 27.0 87.5
7/19 200( 5) 5.0 4.2(1-? ) 43.1 80.0
7/23 150( 1) 0.7 2.0° 43.8 100.0
7/31 100( 3) 3.0 2.7(1-3 ) 58.0 100.0
8/10 115( 0) 0.0 - 30.1 - 00 *0 150( 4) 2.7 2.2(1-5 ) 46.3 100.0
8/22 100( 6) 6,0 2.0(1-5 ) 59.6 100.0
8/29 125( 7) 5.6 2.4(1-4 ) 61.5 80.0*1
9/9 100(12) 12.0 2.0(1-5 ) 43.9 70.0
10/10 50( 0) 0.0 0.0
Totals 1635(93) 5.7 2.5 46.4 83.9
a All flies collected from Herd G during the summer of 1978, Numbers in parentheses indicate number of parasitized individuals in sample. ® One parasitized fly in sample. a One fly also parasitized by Heterotylenchus autumnalis. Fig. 6. Percentage parasitism of female face flies by Thelazia sp. and percentage parous flies collected from Herd G during the summer of 1978. 44 Dd3d AliaVd 1N3 NOI1D33NI !N3Dd3d
oz- 45
Parity and percentage parasitism. The condition of the ovaries with respect to parity is presented in Tables 2 and 3 for 1977 and 1978 sam¬ ples, respectively, and is illustrated in Fig. 6 for 1978 samples. These data indicate that infected flies were most often parous. Of the parasi¬ tized flies sampled from Herds A, B and G in 1977, 74.5> 78.7 and 89.2% were parous compared with 38.6, 32.2 and 47.3% of the total flies sam¬ pled. Similarly, 83.9% of the parasitized flies from Herd G (cf. Table
3) in 1978 were parous compared with 46,4# of the total sample. In addi¬ tion, high percentage parasitism tended to be associated with samples with a high percentage of parous individuals.
Parasite load of infected flies. The range and average number of Thela- zia larvae per parasitized fly is presented for each sample in Tables 1,
2 and 3. Parasite load for all infected flies ranged from 1 to 33 for a total average of 3.1 parasites/fly. The distribution of parasite loads is classified in Fig. 7 into categories of 1 through 10 and greater than
10 parasites/fly. Of 602 parasitized flies 352 (58.6#) carried a load of
1 or 2 nematodes while only 16 (2.6#) contained greater than 10 para¬ sites/fly.
Predilection sites of parasite larvae. The capsules within which Thela- zia larvae undergo early development were found exclusively in the ab¬ domen, attached either to fat body or the body wall. Out of a total of
1,551 capsules observed, 1,215 (78.3%) were found on the body wall, with the remainder attached to fat body (cf. Table 4).
A tendency towards clumping of capsules on either of the attachment Fig. 7. Parasite load of field collected female face flies parasitized by Thelazla sp, in Massachusetts (1976-1978). 250 sand a3io3dNi on
NO. PARASITES PER INFECTED FLY Fig. 7 47 Table 4. Predilection sites of capsule attachment by Thelazia sp. in parasitized female face flies collected from cattle in Massachusetts (197601978).
Abdominal Body tfall Total Capsules Located Om Grand Segments Segment Segment Body Wall Fat Body Total 1 and 2 3 4
1976a _b _b _b 269 92 361
1977 Herd A 124 116 127 367 89 456 Herd B 68 52 63 183 61 244 Herd G 92 50 84 226 62 288
1978 Herd G 61 44 65 170 32 202
Totals 345 262 339 1215(78.3)° 336(21.7)° 1551
f* All samples combined. b Abdominal segment numbers not noted. c Figures in parentheses indicate percentage of total capsules attached to site. 49
Table 5. Distribution of capsule attachment sites of Thelazia larvae in multiply parasitized female face flies collected from cattle in Massachu¬ setts (1976-1978).
1976a 19 77 1978 Herd A Herd B Herd C Herd C Totals15
No. Flies With >1 Capsule/Fly 76 100 51 63 42 332
No. With Capsules On Body Wall Only 48 70 36 42 36 232(69.9)
No. With Capsules On Fat Body Only 15 19 9 11 4 38(17.5)
No, With Capsules On Both Sites 13 11 6 10 2 42(12.6)
^ All samples combined. Figures in parentheses indicate percentage of total multiply parasi¬ tized flies belonging to class. 50 sites in multiply parasitized flies was observed (Table 5) • Of 332 flies containing more than 1 capsule, 232 (69.9%) bad capsules attached only to the body wall while 58 (l7*5%) bad all capsules attached to fat body.
Only 42 (12,6^6) of the multiply parasitized flies possessed capsules at¬ tached to both predilection sites.
The number of parasitized flies with greater than 1 nematode/fly exceeds the number of flies with greater than 1 capsule/fly. This is due to capsule fragmentation by departing invasive larvae and because, on occasion, single capsules were found to contain more than one nema¬ tode.
Discussion and Conclusions
Geographic range. Results of the 1976 statewide survey of female face flies indicate that bovine thelaziasis is both present and widespread in Massachusetts, In addition, the overall incidence of parasitism found during this survey (2.6$) is considerably higher than that observed
(.95%) in a similar survey, restricted to two counties in the western part of the state (Branch and Stoffolano, 1974), It is difficult to ascertain whether this increase reflects a true increase in the preva¬ lence of thelaziasis or merely the chance sampling of more heavily in¬ fected herds in the present study.
Temporal patterns of parasitization. During 1977 and 1978 sampling, a similar, bimodal pattern of parasitization was observed in the face fly populations associated with the study herds; an early summer peak, a mid¬ season depression, and a late summer peak. Two factors may account for 51
such a bimodal pattern. First, the population dynamics of the fly, more
specifically, the age structure of the flies feeding on the host and
hence inclusion in the samples, and, second, the population dynamics of the nematode parasite, i.e. seasonal changes in the number of sexually mature, adult female eyeworms. In an attempt to assess the influence of the first factor, parity data was obtained for most 1977 and all 1978
fly samples, A relationship between high percentage parasitism and high percentage parous females was observed to be strongest in the early sea¬
son samples, the association being less clear later in the summer. Parity gives an estimate of the age structure of the fly population, dividing the sample roughly into classes of less than six day old (nulliparous), and greater than six day old (parous) individuals (Miller and Treece
1968b). Such a classification, however, masks real age differences among uni-, bi-, and triparous flies, and thus is not a completely reliable
indicator of fly age.
Other evidence indicates that the bimodal pattern may be at least
partially due to changes in the age structure of the nematode population.
Soviet workers have reported that large numbers of overwintering, adult
parasites are available for early summer infections of the vector, but
that the adult population crashes by the end of June, with most of the
infections in July and August consisting of juvenile forms (Klesov, 1949a,
1953). Similarly, Arbuckle and Khalil (1978), in England, found that the
percentage of adult eyeworms in infected bo vines declined steadily in
June and July, increasing again in August, September and October. Of the
adults found in the latter part of the season, over 99% had developed
from larvae established in hosts earlier in the summer. Since the pre- 52 patent period for Thelazia eyeworms is between 45 days and two months
(Klesov, 1949a, 1953? Krastin, 1949, 1958; Ivashkin, 1953) the two peaks of parasitization in the flies may be a reflection of the overwintering and new generation adult nematode populations in early and late summer, respectively, with the mid-season depression occurring when predominant¬ ly immature parasites are present in their bovine hosts.
Vector potential of house fly. Gupta (1970) reportedly found gulosa - in house fly in India, however, the parasite designation was uncertain and was based on a singly fly. In a survey of the flies associated with cattle in Czechoslovakia, Vilagiova (1964) reported house fly to be pa¬ rasitized by rhodesii and gulosa. Unpublished, preliminary experi¬ ments by these authors indicate that, in infections of colony flies, T, gulosa successfully penetrates the midgut of M_j_ domestica and develops abnormally, free in the hemocoel, for up to 9 days before melanization occurs. These data, in addition to field studies presented in this paper, indicate the unsuitability of the house fly as a vector of eyeworms in
Massachusetts.
In other insect-nematode systems, vector suitability has been dem¬ onstrated to be under genetic control. McGreevy et al. (1974), testing the susceptibility of 9 strains of Aedes aegypti (Linnaeus) to parasiti¬
zation by Dirofilaria immitis (Leidy), found 3 completely refractory
strains, 5 strains of intermediate susceptibility and 2 that were 86-
89% susceptible. Similarly, experimental infections of different strains
of Aedes aegypti and Ae. togoi (Theobold) with Wuchereria bancrofti (Cob-
bold) showed refractory and susceptible strains of each species to the 53
parasite (Partono et al., 1977). Susceptibility within a vector species
has been demonstrated to be governed by a single, recessive, sex-linked
gene for Culex pipiens Linnaeus to Brugia pahangi (Buckley and Edeson)
(Obiamwe and MacDonald, 1973) and Aedes aegypti to Dirofilaria immitis
(McGreevy et al., 197*0. On the other hand, Lawrence and Pester (1967),
in a series of infections spanning 4 generations of the vector, conclu¬
ded that the increasing susceptibility of Aedes togoi to Brugia patei
over time was due to selection pressures on the parasite, rather than
the genetics of the vector. Perhaps, in parts of the world where house
fly and Thelazia sp. have had a longer association, the fly has evolved
into a competent vector of eyeworms.
Parasite load of infected flies. The number of immature Thelazia sp, per
infected face fly was found to be fairly low (3.1), and showed no corre¬
lation with percentage parasitism. Dirofilaria immitls incurs a consid¬
erably greater worm burden in its culicine hosts. In experimental infec¬
tions, Kartman (1953) found an average parasite load of 18.1 and 19.8 •4 Et. immitis per infected Anopheles guadrimaculatus Say and Aedes alboplc-
tus (Barraud), respectively, while Christensen and Andrews (1976) found
an average of 8.6 nematodes per infected, field-collected Aedes trlvatta-
tus (Coquillet) in Iowa. Hairston and DeMeillon (1968) reported a para¬
site load of 5.0 Brugia malayi (Brug) per infected Anopheles anmii.+.«,
DeRook in East Pahang and 4.5 Wuchererla bancroftl per parasitized Culex pipiens fatigans tfiederaan in Rangoon. In a survey of black flies from four villages in Cameroon, Duke et al. (1972) found the mean number of
Onchocerca volvulus (Leuckart) per parasitized Simulium damnosum Theo- 54
bald to range from 1.6 to 2.3. Parasite load in a vector species varies
considerably from one location to another, depending on the susceptibi¬
lity of the local vector and the infectivity of the parasite. Because of
the epidemiological importance of parasite load to transmission poten¬
tial, greater attention should be paid to this aspect of Thel*?fla in the
intermediate host in future research.
Predilection sites of parasite larvae. At present, there exist no relia¬
ble diagnostic characters for species determinations of immature eye-
worms in the pre-invasive stages. Our data on capsule attachment sites
suggest that these sites may be species specific, as indicated by the tendency towards clumping of capsules on only 1 site in 8?.<4^ of mul¬ tiply parasitized flies. Further, data on experimental infections of laboratory-reared face flies have indicated that Thelazia gulosa larvae become encapsulated almost exclusively on the body wall of the vector
(this thesis, Chapter IV). The 12.6* of multiply parasitized flies with capsules attached to both predilection sites may be due either to flies having visited 2 bovines parasitized by different Thelazia species, or to their having fed around the eyes of an animal harboring both species of parasite.
Vilagiova (1967) reported both Tj_ gulosa and skrjaMm to deve¬ lop within the egg follicles of infected female autnmnans. The re¬ sults of this study did not confirm that observation; of 602 parasitized flies examined, none were found to possess parasites developing in that fashion. CHAPTER III
BOVINE THELAZIASIS IN MASSACHUSETTS i FIRST
REPORT IN THE NORTHEASTERN UNITED STATES.
Introduction
The genus Thelazia Bose consists of a cosmopolitan group of nema¬
tode parasites of the eyes of mammals. Until the beginning of the pres¬
ent decade the genus was thought to be represented in North America by a
single, endemic species, Thelazia califomiensis Price, a parasite of
the eyes of a number of mammals (Knapp et ail., 1961). The range of this
species has been primarily restricted to the western and southwestern
United States (Beitel et aJL., 1974),
In 1970 an equine eyeworm, Thelazia lacrymalis (Gurlt), new to the
continent but prevalent in the Old World, was discovered in the eyes of
horses in Maryland (Walker and Becklund, 1971) and Ontario (Barker,
1970). A survey of dead horses in Kentucky (Lyons and Drudge, 1975b) re¬
sulted in the realization that this eyeworm may be far more prevalent
than previously thought, with 26.4# of the animals proving positive for
thelaziasis.
While dissecting female face flies, Musca autumnalls DeGeer, colle¬ cted from a herd of dairy cattle in western Massachusetts in 1970, Chit¬ wood and Stoffolano (1971) discovered an individual fly parasitized by
Immature Thelazia sp,. Because the face fly is a known vector of several species of bovine eyeworms in Europe (Vilagiova, 1964), and of T\ iacry_
Sails in the U.S. (Lyons et al., 1976), follow-up surveys of fly popu-
55 56
lations associated with cattle in Massachusetts were conducted. The re¬
sults of these studies (Branch and Stoffolano, 197^» this thesis, Chap¬
ter II) indicated that bovine thelaziasis was not only present but wide¬
spread in its distribution in this state. The dearth of adequate taxono¬
mic information on the immature forms found in field-collected flies,
however, precluded the possibility of species determinations.
Surveys of the eyes of dead cattle in Kentucky (Lyons et al., 1975»
Lyons and Drudge, 1975a) revealed the presence of two bovine eyeworms,
Thelazia gulosa (Raillet and Henry) and T^ skrjabini Erschov, providing
the first documented reports of bovine thelaziasis in North America.
These workers found eyeworm infections to be almost entirely subclinical.
Abattoir surveys in England (Arbuckle and Khalil, 1976, 1978j Turfey and
Chandler, 1978), during which both T\ gulosa and T\ skr jabini were found,
also indicated the predominance of subclinical infections, with lesions
present in a small percentage of infected eyes. In other parts of the
world, however, the same species have been reported to commonly produce
symptoms which include profuse lacrimation, mucopurulent discharge, conjunctivitis and, less frequently, corneal opacity (Chauhan and Pande,
1973)» implying that local factors may play a role in the expression of pathology.
Because of the potential veterinary hazard presented by Thelazia eyeworms and our uncertainty of the species present in the Northeast an abattoir survey of bovine eyes in Massachusetts was conducted to deter¬ mine (1) the species of Thelazia present* (2) the incidence of eyeworm infections; and (3) the extent and prevalence of pathology associated 57
with such infections.
Materials and Methods
Two abattoirs were visited between February of 1977 and. September of 1978. Eyes were immediately removed after slaughter by making a comp¬ lete incision in the hide about 4 cm from the lids and removing the en¬ tire ocular apparatus, including the eyeball itself plus the lids and lachrimal glands. Eyes were then placed, unpaired, in an ice chest until a sufficient amount of material was obtained, at which time they were returned to the university for examination.
Eyes were inspected for parasites by first everting the eye through the lids and examining the exposed surface of the conjunctiva, the con¬ junctival. sac and the hide around the lids. Manual pressure was then ap¬ plied, from within, to the lachrimal ducts of the lower eyelid, which everted the ducts (Fig. 8) and forced out nematodes lodged within. The third eyelid was then either turned back or excised (Fig. 9)» and pres¬ sure was applied in a similar fashion to the ducts of the nictitating membrane glands. Fig. 10 shows a mature gulosa eyeworm laid across a bovine eye to give an indication of the size of the parasites relative to the eye.
All nematodes recovered from an individual eye were placed collec¬ tively in a Syracuse watch glass flooded with physiological saline (.85%
NaCl) and placed alongside the infected eye. Parasites were counted,
identified, and, where possible, sexed. Sexually mature female eyeworms
were kept alive for experimental infections of laboratory-reared flies
(this thesis, Chapter IV); the remainder were placed in fixative (FAA) Fig. 8. Bovine eye everted through lids to illustrate location of lachrimal ducts, preferred site of Thelazia gulosa.
Fig. 9. Bovine eye everted through lids to illustrate location of third eyelid. skrjabinl is most often found in ducts originating at the base of this structure.
Fig. 10. T_j_ gulosa parasite laid across a bovine eye to illustrate size of the nematode relative to the eye.
Fig. 11, Abscess on surface of conjunctiva from which a nodule of gulosa worms was removed.
62 for future measurements. Infected eyes were macroscopically examined for lesions and other overt, post-mortem pathological indicators (Fig. 11).
Results
Species of "bovine Thelazia. Two species of bovine eyeworms, T^_ gulosa and Tj_ skrjabini, were identified. Thelazia gulosa is characterized by the deep, cup-shaped buccal capsule (Fig. 12), the relatively pointed posterior end of the female (Fig. 13) and the greatly dissimilar spicule lengths of the male (Fig, 16), T^ skr jabini is characterized by its smaller buccal capsule which is narrower at the anterior end than at the base (Fig. 14), the more blunt tail of the female (Fig. 15) and the sub¬ equal spicules of the male (Fig. 17). Measurements of preserved speci¬ mens are presented in Table 6? however, it should be noted that these measurements represent the lower end of the scale since older, sexually mature adults were sacrificed for experimental purposes and hence are not included in the sample.
Incidence of infection. Of 795 eyes examined during the survey 97, or
12.^6 were found infected with Thelazia sp. Seventy-four of the infec¬ ted eyes were parasitized by 1\ gulosa and 8 by T^ skr jabini alone, with
15 showing mixed infections of both species. gulosa was found most frequently in groups in any one of the lachrimal ducts of the lower eye¬ lid and, less commonly, in the ducts at the base of the third eyelid. T. skr jabini was found more often in the latter location. Both species were also found in the conjunctival sac, wandering across the conjunctiva or l11 the hide immediately surrounding the eye. 63
Fig. 12, Thelazia gulosa, anterior end of female. (1 in = .049).
Fig. 13. Thelazia gulosa, posterior end of female. (1 in = ,049 mm).
Fig. 14. Thelazia skrjabini, anterior end of female. (1 in = .049 mm).
Fig. 15. Thelazia skr jabini, posterior end of female. (1 in * .049 mm).
be * buccal capsule 64 65
Fig, 16. Thelazia gulosa, posterior end of male, (1 in = ,196 mm).
Fig. 17. Thelazia skrjabini, posterior end of male. (1 in 3 ,196 mm).
sp = spicules 66 67
Table 6. Measurements of adult bovine Thelazia worms from a Massachu¬ setts abattoir survey (1977-1978).
T, gulosal T, skrjabini^
Male Female Male Female
Body Length (mm)3 7.1-8.6 7.5-12.9 - 7.5-10.4 n.6-15.0 Width At esophageal bulb 104-160 104-148 80-118 96-124 Maximum 190-360 241-304 171-209 171-240 At anus or cloaca 57-98 57-93 72-86 57-95 Buccal capsule Width Anterior end 31-39 31-44 14-22 13-20 Base 19-22 21-32 12-22 18-25 Depth 21-28 21-30 5-11 6-12 Esophagus Length 218-291 242-323 294-357 304-388 Width at bulb 48-62 55-86 43-57 48-63 Nerve ring from anterior end 148-185 156-240 190-238 304-388 Vulva from anterior end — 390-551 — 486-659 Anus or cloaca from anterior end 57-70 57-114 48-66 Spicule length 67-95 Small 126-147 96-108 Large 610-780 — 108-145 ___
2 From 20 females and five males. From 13 females and five males. All other measurements in microns. 68
Parasite load of Thelazla infections. Thelazia skr jabinl was the lesser
abundant of the two species, with the number of nematodes per infected
eye ranging from 1 to 5 with an average of 1.8. A total of 42 skrja-
bini (31 females, 8 males, and 3 undetermined specimens) were recovered.
T, gulosa was present in greater numbers, with a parasite load ran¬ ging from 1 to 81 with an average of 18.4 nematodes per infected eye. Of the 1,640 gulosa found, 1,168 were females, 431 were males and 41 were undetermined.
The number of parasites associated with all Thelazia infections is presented in Fig. 18. Of 97 infected eyes 52 (53.^6) carried 10 or fewer nematodes while only 8 (81.3^) carried a burden of over 50 parasites per infected eye. V
Pathology associated with Thelazia infections. Two of the infected eyes, both parasitized by gulosa alone, displayed pathology, either due to or associated with the parasites. In one case a nodule of 31 nematodes was found just under the surface of the conjunctiva, accompanied by slight general conjunctivitis, and extensive lesions with rather intense vascular congestion in the immediate area of the nodule (Fig. 11).
In the second case, the pathology was much reduced. A mass of 13 juvenile gulosa worms was recovered from the conjunctival sac, with accompanying slight scarification, localized edema and redness.
No correlation between eyeworm infections and corneal lesions or opacity was made. Fig. 18. Parasite load of Thelazia eyeworms observed in bovine eyes from Massachusetts aba¬ ttoirs. 70 NO. PARASITES PER INFECTED EVE
S3A3 G31D3JNI ON
i 71
Discussion
Adult bovine and equine eyeworms of the genus Thelazia require in¬ termediate hosts of the dipteran genus Musca for their development. The fly ingests first instar larval nematodes released by adult parasites into the lachrimal secretions of the host. After a developmental period of about 10 days in the hemocoel of the fly, the third instar invasive stage larvae migrate to the head of the fly and exit through the probos¬ cis while the fly feeds about the eyes of a host. The parasites then mi¬ grate to their predilection site within the ocular apparatus, develop to the adult stage, mate and begin to produce a new generation of larvae.
The close association which the face fly, Musca autumnalis, maintains with horses and cattle renders it an ideal agent for the vectoring of eyeworms. The arrival of the face fly onto the North American continent in the early 1950*s (MacNay, 1952) made continuous transmission of eye- worms possible for the first time, since the only other Nearctic Musca species, the house fly (M^ domestica L.), appears to be an unsuitable intermediate host for Thelazia sp. (this thesis, Chapter II).
The discovery of adult eyeworms in Massachusetts confirms earlier work with field-collected face flies in the state (Branch and Stoffo- lano, 1974; this thesis, Chapter II) and represents the first report of bovine thelaziasis in the northeastern U.S. The present study, as well as others in Kentucky (Lyons and Drudge, 1975a) and Ontario (Surgeoner and Moolenbeek, pers comm.) would indicate that these eyeworms are well established within the range of the face fly.
Thelazia gulosa and skrjabini have an extensive distribution in 72
the Old World. Both species are present throughout much of the Soviet
Union (Skrjabin et al., 196?), Eastern Europe (Vilagiova, 196?; Dulcea-
nuf 1971)» Germany (Eckert et al., 1964), England (Arbuckle and Khalil,
1976), India (Chauhan and Pande, 1973) and Japan (Okoshi and Kitano,
1966a,b). In addition, T_j_ skrjabinl has been reported from Denmark (Kol-
strup, 1974) and gulosa from Israel (Franzos, 1964).
The method by which the parasites were introduced into the United
States is not known. They may have been imported with a shipment of in¬
fected livestock or accidentally introduced with the face fly. Another
possible route of entry is through the practice of importing exotic al¬
ternate hosts for American zoos. Thelazia gulosa was first found in the
U.S., not in a domestic bovine, but in an imported giraffe (Walker and
Becklund, 1971). It is interesting, from the standpoint of exotic dis¬
ease introduction, that the giraffe was quarantined twice, once in Afri¬ ca and once in Clifton, N. J., before being placed in the zoo. The ship¬ ment of cattle at the intra- and international levels promotes the in¬ troduction of eyeworms from endemic zones into uninfected areas. Our current inability to make ante-mortem diagnoses of thelaziasis poses the threat of introduction of Thelazia rhodesii, a highly pathogenic eyeworm prevalent throughout Asia, Africa and Southern Europe, which also can be transmitted by face fly (Skrjabin et al., 1967).
During the present study, the incidence of thelaziasis was relati¬ vely low (12.^). This figure represents the percentage of total eyes, ajid not the percentage of animals, infected. For this reason the present incidence data may appear misleadingly low. Lyons and Drudge (1973a) re- 73
ported an Infection rate of 15$ of the animals surveyedj however, since
the percentage of bilateral infections was low (11.5$ of infected ani¬
mals), the percentage infection of total eyes was considerably lower
(8.2$). Because of the limited mobility of eyeworms and their inability
to withstand dessication, the frequency of direct eye-to-eye transmis¬
sion in individual hosts is probably negligible, with bilateral infec¬
tions being largely a probability function dependent on the number of
parasites available for infection in the vector population. For example,
Arbuckle and Khalil (1978) found an incidence of infected animals of
41.9$. When the data are translated into percentage infected eyes, a rate of 28.8$ is derived. Because of the higher percentage of infected
eyes, an accompanying higher percentage of bilateral infections was ob¬
served, i.e. 3%% of the infected animals. This figure is somewhat higher than might be expected if individual eye infections were completely in- dependent events. This may be attributed to multiply parasitized flies discharging nematodes into both eyes during a single visit to a host.
Also, animals already infected in one eye may be more attractive to the flies than uninfected hosts by virtue of the excessive lacrimation issu¬ ing from the infected eye.
Pathology was observed in only 2 of 97 infected eyes. Pathogenesis associated with Thelazia gulosa and T\ skrjabinl has been observed to be mild, rare or absent in the U.S. (Lyons and Drudge, 1975a), Germany
(Eckert et al., 1964), England (Arbuckle and Khalil, 1978) and Japan
(Okoshi and Kitano, 1966), On the other hand, common symptoms ranging from conjunctivitis through corneal lesions to blindness have been att- 74
ribated to these parasites in Mongolia (Ivashkin, 1953)» Southern Russia
(Gorodovitch, 1963), India (Chauhan and Pande, 1973) and, for gulosa only, Israel (Franzos, 1964). The almost complete lack of clinical sym¬ ptoms in Europe, Japan and the U.S. may be due to differences in live¬ stock management, climatic factors or to strain differences among para¬ site and host species. At present, nothing is known about the factors governing the expression of pathology in this group of eyeworms.
Conclusions
The discoveries in recent years of surprisingly high rates of bo¬ vine and equine thelaziasis in the U.S. underscore our lack of infor¬ mation about this group of eyeworms. While Thelazia infections in this country have been found to be predominantly subclinical on post-mortem investigation, we have yet to assess the economic injury which may re¬ sult from decreased beef and dairy production in infected herds. The re¬ ported symptoms of bovine thelaziasis parallel those of infectious bo¬ vine keratoconjunctivitis (Stoffolano, 1970). Major obstacles to such an assessment will be the discrimination between the numerous etiological agents of IBKC (Bedford, 1976; Morgan, 1977) and the development of ef¬ ficient ante-mortem diagnostic techniques for eyeworms. Further, should thelaziasis come to be regarded as an important disease of livestock, an appropriate anthelminthic must be registered or developed. At present no such drugs are registered for the treatment of eyeworms on the domestic market. Future priorities in Thelazia research should be directed to- 75 wards these three areas of diagnosis, assessment of importance, anrf treatment. CHAPTER IV
DEVELOPMENT OF THE BOVINE EYEWORM, TFET.AZTA GULOSA
(raillet and henry) in experimentally infected,
LABORATORY-REARED, FEMALE MUSCA AUTUMNALIS DEGEER
Introduction
In recent years, 3 species of Thelazia eyeworms not previously re-
ported on the continent have been reported in North American horses and
bo vines. Thelazia lacrymalis (Gurlt) has been recovered from the eyes of
horses in Ontario (Barker, 1970), Maryland (Walker and Becklund, 1971),
Kentucky (Lyons and Drudge, 1975b), and Quebec (Frechette et al., 1976),
while the bovine eyeworms Tj_ gulosa (Raillet and Henry) and T\ skr.jabinl
Erschov have been identified in cattle in Kentucky (Lyons and Drudge,
1975a) and Massachusetts (this thesis, Chapter III),
Most eyeworm research has been conducted outside the U.S., and has
focused primarily on surveys of adult parasites in the definitive verte¬
brate host, therapeutic evaluations, and regional sampling and assess¬
ment of local potential vector species, nearly all of which are filth
flies of the genus Musca (Stoffolano, 1970). Experimental studies on the
development of Thelazia sp. in the intermediate host have seldom been
conducted. The bovine eyeworm T\ rhodesii Desmarest has been studied in teca larvipara Portchinsky (Klesov, 1950), fu convexifrons Thompson
(Skrjabin et al., 1967), autumnalls DeGeer (Vilaglova, I967), and
~ Villeneuve (Miyamoto et al., 1974). gulosa was studied by
Austin (in Skrjabin et al., 1967), in M^ arnica Zimin and by Vilaglova
76 77
(1967) in M#. autumnalis. Kozlov (in Skrjabin et al., 1967) observed the
development of callipaeda Rail let and Henry, an eyeworm of dog, cat
and man, in Phortica (Amiota) variegata (Fallen), while Dobrynin (1974)
studied the camel parasite, T\ leesi Raillet and Henry, in Musca lucidu-
la Leow. Only one study (Vilagiova, 1967) has dealt with the development
of T± gulosa and 1\ skrjabini in their Nearctic intermediate host, the
face fly (M^ autumnalis),
Several observations reported in the study by Vilagiova prompted a
reexamination of the relationship between these parasites and the face
fly. First, she observed that gut penetration of the fly did not occur
until 24 hr post-infection, an observation which differed from those made
by Klesov (1950) and Kras tin (in Skrjabin et al., 196?) and, in relation
to other insect-nematode systems, seemed rather long. Second, she found
that Thelazia skrjabini did not reach the invasive 3rd stage in face fly
within 32 days, and that gulosa required 28-32 days to develop to the
3rd stage larvae. Casual observations made during a face fly survey in
Massachusetts (this thesis, Chapter II) suggested that development re¬
quired considerably less time. Third, she reported that the capsules
within which immature nematodes develop were located in the egg follic¬
les of female flies; however, in Massachusetts, all capsules were found
attached either to the abdominal body wall or to fat body.
The present study had two objectivess first, to determine the time
and location of gut penetration of gulosa in experimentally infected,
laboratory-reared autumnalis; and, second, to observe the development of this parasite to the invasive stage in the fly. 78
Materials and Methods
Adult T^ gulosa worms were obtained from bovine eyes during an eye- worm survey of Massachusetts abattoirs. Techniques for eye removal and parasite recovery are described elsewhere (this thesis, Chapter III).
Sexually mature female nematodes (Fig. 19) were individually placed in depression slides with a few drops of physiological saline (Norman and
Duve, 1969). The head of the parasite was severed with forceps at a point just anterior to the vulva, with the sudden release of hydrostatic pressure resulting in the expulsion of the anterior portion of the ova¬ ries. The uterus was then severed and the ovaries pulled away from the body. The anterior portion of the ovaries plus the uterus were then carefully dissected to liberate recently hatched nematode larvae (Fig.
20). Using suction, parasite larvae were taken up into a drawn capillary tube affixed to 6 cm of polyethylene tubing. Drops of saline containing larval nematodes were then presented to restrained, 5-7 day old, female face flies which had been deprived of all food and water for 12 hr.
Flies accepting the parasite-saline solution were subsequently presented ad lib. with warm, citrated beef blood; those refusing the infective sa¬ line were discarded.
For the gut penetration studies, the entire alimentary tracts of flies were dissected out l/2 hr post-infect ion and placed, in groups of
3, in depression slides flooded with physiological saline (cf. above).
These were then placed under a dissecting microscope at 25x and scanned
^til nematodes began to emerge through the gut wall. The onset, dura¬ tion, and location of gut penetration were noted for each fly. Fig. 19. A portion of the anterior region of a sexually mature female gulosa parasite showing ovaries packed with nematode larvae, (l in = .196 mm).
Fig. 20. A recently hatched gulosa larva. Arrow indicates egg shell clinging to posterior end. (1 in * .049 mm).
81
For the development studies, recently infected flies were placed
into cages, provided with granulated sucrose, powdered milk, bovine man¬
ure, c it rated beef blood and distilled water. Cages were kept in a
growth chamber maintained at 18 hr Lj6 hr D photoperiod, 28-30°C, and
6C$ RH, Parasite load and capsule attachment sites were recorded for
"lies dissected 3> 6 and 9 days post-infect ion. Nematodes were placed in
warm fixative (FAA) and later cleared in phenol for measurements. No
assessment of morbidity or mortality in the flies was made.
Results
t, penetration. The alimentary tracts of 24 experimentally infected
face flles were examined for penetration by gulosa larvae. Of these,
7 were successfully penetrated during the 5 hr observation period comm-
!cing l/2 hr after the infective saline meal. Larvae were always obser-
ad to Penetrate the mid-gut, a process which ensued 1-2 hr post-infec-
‘ ion. Penetration continued for up to 4 hr post-infect ion. By 5 hr, all
remaining larvae were found in the hind-gut. No record was kept of the precise location of, or of the time required for the actual act of gut penetration by individual larvae.
Development of T, gulosa In M. autumnalls. A total of 137 female face
Hies were fed on T\ gulosa larvae, of which 26 died before dissection,
63 were successfully parasitized and 48 were uninfected. An average pa¬ rasite load of 2.1 nematodes/infected fly was observed, with a range of
1 fo 18. All capsules were attached to the abdominal body wall except for 2 adhering to fat body in a fly with 18 parasites. 82
Measurements of 15 parasites on 3> 6 and 9 days post-infection are
presented in Table 8.
On day 3» all live nematodes were found in capsules attached to the
body wall. In 3 flies containing live encapsulated larvae, dead, non-en-
sapsulated larvae, which resembled newly hatched forms, were found free
in the hemocoel. The encapsulated larvae, at this stage of development,
were largely undifferentiated (Fig. 21); the only visible internal
structures were the poorly defined esophagus and rectal, ampule. The
buccal capsule, nerve ring, esophageal bulb, and cuticular striations
ware undefined. Width was greatest at the anus, gradually decreasing an¬
teriorly. Total length ranges from 225 to 297 microns (Table 8).
3y day 6, the molt to the second instar had occurred. All structu-
■ ss, except for rudimentary genitalia and striations, were clearly disc- ernable (Fig. 22). The nerve ring was located at a point roughly half the length of the esophagus, which comprised 15-2Q£ of the total body length. The buccal capsule had assumed the cup-like appearance of inva¬ sive larval and adult forms. The rectal ampule was roughly twice as long as wide at its widest point. Larvae were broadest at, or just posterior to, the esophageal bulb. Total length ranged from 528 to 776 microns
(Table 8).
By day 9, the 2nd molt to the 3rd instar had taken place. Larvae were found moving actively within the capsules or free in the abdomen
thorax. Striations were clearly visible (Fig. 23) and appeared, in the cephalic region, in numbers of 11.4-15.8 per 100 microns. The nerve
tag, located 1/2-2/3 the length of the esophagus, comprised ca, 10^ of 83
total body length. In females the vulva was visible, and located 282-346
microns from the anterior end. Rudimentary spicules could usually be
discerned in males. The rectal ampule had increased little in width but
was twice its day 6 length. (Fig. 24). Body width was greatest in the an¬
terior one-third of the parasite, generally just posterior to the eso¬
phageal bulb. Total length ranged from 2.1 to 2,3 mm.
Discussion
The larvae of Thelazia gulosa were observed to initiate penetration
of the mid-gut of female autumnal is within 1-2 hr post-infect ion, and
to continue penetration for up to 4 hr, after which the larvae were found
in the hind-gut. This observation differs markedly from that made by Vi-
lagiova (1967), who found active larvae in the gut as late as 24 hr post¬
infection. Klesov (1950) found penetration of Musca larvipara by T. rho-
desii to occur within "several hours" of infection; however, no details
were given.
The method of gut penetration by Thelazia larvae is not known. The immature nematodes appear to possess neither stylets nor any apparent specialized morphological adaptations for penetration, with the excep¬ tion of the larvae of callipaeda (Skrjabin et al., 1967), which have
Posteriorly pointing spines on the cuticle to facilitate forward move¬ ment through the tissue. Digestive enzymes may play a role in this pro¬ cess.
In the present study, development of gulosa to the invasive st*ge larva was completed in 9 days. Vilagiova (1967) found such deve- 84
. „ 2^' li SHi22| larva from face fly at 3 days post infection. (1 in = .196 mm).
Fig. 22. Tj_ gulosa larva from face fly at 6 days post infection. (1 in = .196).
an - anus, be = buccal capsule, eb = esophageal bulb, nr = nerve ring, ra = rectal ampule.
Fig. 23. Anterior end of T^_ gulosa invasive larva from face fly at 9 days post-infection. (1 in * .196 mm).
Fig. 24. Posterior end of gulosa invasive larva from face fly at 9 days post-infection. (1 in = .196 mm).
an = anus, be * buccal capsule, cs ~ cuticular striations, eb =* esophageal bulb, nr = nerve ring, ra * rectal ampule. 87 88
Table 7. Measurements of key morphological features of Thelazia gulosa in experimentally infected, laboratory-reared female face flies (Musca autum- nalis.
Days Post-Infection . 3 6 9 range (mean) range (mean) range (mean)
Body Total Length (mm)a .225-.297 (.253) .528-.776 (.708) 2.120-2.5^0 (2.3) Width Esophageal Bulb13 14-25 (19) 69-83 ( 81) 80-101 ( 92) Maximum 22-45 (29) 71-92 ( 82) 89-110 ( 97) Anus 22-45 (29) 41-55 ( 50) 44-55 ( 48) Buccal Capsule Width Anterior End — 8-12 ( 9) 12-18 ( 14) Base — 7-10 ( 8) 7-12 ( 8) Depth - 5-7 ( 5) 7-14 ( 11) Esophagus $ 00 VO Length 1 (51) 97-133 (124) 218-245 (235) Width at Bulb - 25-34 ( 3D 44-60 ( 55)
Distance to Nerve Ring - 60-81 ( 71) 127-146 (137)
Distance to Vulva - - 282-346 (315) Distance to Anus Prom Posterior End 36-45 m 34-55 ( 47) 62-80 ( 75) \ Rectal Ampule Length 9-14 (12) 60-74 ( 70) 136-173 (153) Width (Max.) 9-U ( 9) 32-41 ( 36) 27-45 ( 40) ffpt Striations0 - 11.9-15.8 Tooj: (13.1]
b All other measurements in microns. c Position of esophageal bulb estimated for day 3 larvae. s+-nDf^erMined ^ measuxinS the distance from the first visible anterior -nation to the 20th, followed by the appropriate conversion. 89
lopment to require 28-32 days. In related studies, T^ rhodesii developed to the 3rd instar in 15-30 days in larvipara (Klesov, 1950), ca. 30 days in convex ifrons (Skr jabin et al., 1967) and 28-32 days in au- tumnalis (Vilagiova, 1967). Dobrynin (197*0 found the molt to the 3rd instar of leesi in lucidula to occur on day 10-11, while Kozlov
(in Skr jabin et al., 1967) found T_j_ callipaeda in Phortica variegata to require 19-22 days. Lyons et al. (1976) observed lacrymalis larvae to attain the invasive stage in M^ autumnalis in 12-15 days.
The differences in developmental time observed during this study and in that of Vilagiova (1967), using the same parasite and host, axe rather striking. Much of this disparity probably arises from differences
In technique. Vilagiova, for example, used second generation field-coll¬ ected flies for her experimental infections, while we used an establish¬ ed fly colony obtained from a stock culture at Beltsville, Maryland. , Also, fly age, nutrition, and environmental conditions may have pro¬ found influences on the rate of development of Thelazia larvae in the vector.
The parasite load noted in this study was lower (2.1) than that ob¬ served (3.1) in naturally infected, field-collected flies (cf. Chapter
II). This also may be attributed to technique. The physiological readi¬ ness of larvae dissected from the ovaries of adult eyeworms to penetrate and become established in the fly may not be as great as that of larvae actually expelled from females in the lachrimal secretions of the defi¬ nitive host under natural conditions.
Similarly, the rather low success rate of parasitism in the flies 90
(5^*3%) is likely more a reflection of infection technique than of in¬
herent factors in the host-parasite relationship. In the initial infec¬
tions, the capillary tube into which the larvae were drawn was filled
with sufficient parasites to infect 5~10 flies at a time. This process,
however, resulted in many of the nematodes sinking to the lower end of the tube, producing a low success rate of parasitism (10-15^). By shift¬
ing to individual fly infections, the success rate increased to 70-85%.
Conclusions
Much remains to be done on the juvenile development of Thelazia eyeworms in the intermediate host. Major gaps in our understanding of the life cycle of these parasites include the mechanism of gut penetra¬ tion and the origin, function, and ultrastructure of the capsules. More¬ over, due to the present lack of effective ante-mortem diagnostic tech¬ niques for adult eyeworms in the definitive host, vector sampling re¬ mains an important means of determining the presence of thelaziasis in
■uspected cattle herds. Given the pronounced differences in pathogenici- ty among bovine eyeworm species, species determinations in the juvenile stages in the intermediate host may become imperative in the event of the introduction of other, more pathogenic, species into North America.
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