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This is to certify that the
thesis entitled

An Entomological and Epidemiological study
of Equine Onchocerciasis in Michigan

presented by
Willie James Roberts

has been accepted towards fulfillment
of the requirements for

Master of Science degeehi Entomology

W

Major professor

Dateihifmw

0-7839 MS U it an Waive Action/Equal Opportunity Institution

 

 

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ANEINITONDIDGCALAADEPIEBMOGCALSTIDYCTEQLDE
OBDHOCERCIASISINMCHIGAN

By

Willie James Roberts

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

NIASTERGSCB‘CE

Department of Entomology
1 986

l

.3

42a 33‘

ABSTRACT

AN ENTOVUIBCALABD EPWDALSTLDYO: ECUIE
WRCIASIS IN MCHIGAN

By

Willie James Roberts

A field study was conducted to evaluate the role of W,
particularly Q. 91159131113 (Meigen) as potential vectors of W filarial
nematode infections in Michigan equines. Qyjjggigefi were collected at the study
site using a blacklight trap and from the infected equines by aspirator.
Quljgmgesgbsgjems.Q.1.1amD_e_nnis (Coquillett), and Q. sielliier (Coquillett)
ingested microfilariae after feeding on an infected horse. These three species
were also found naturally infected with Qnmggema type larvae. In experimental
infection studies, wild caught Q. omelet“: and Q. 1. yafijnennjs were allowed to
feed on an infected horse. Second stage filarial larvae were found in the
thoracic muscles of Q. M91145 2 and 11 days post feeding and in Q. 1.
mm 6, 7, 8, 10, 11, 18. and 20 days after feeding. Qngnggema type
infective stage larvae were found in the head of wild caught and laboratory
reared Q. 1. yarijpennis, 6 and 25 and 23 days, respectively, after feeding on an
infected horse. It is concluded that Q. y. yanipennjs may be a potential vector of
equine onchcerciasis in Michigan and that Q. slfilll'lfl may be a secondary
potential vector. The role of Q. mm as a potential vector is still
unresolved, since no Qnghggema infective stage larvae were found in several

hundred dissections of wild caught midges.

To Teresa

I would like to express my sincere appreciation to the equine owners;
Cindy Jones, Robert F. Bunn, Wayne K. Ross, and Marcy Fisher for the use of
their horses and ponies during this study. I would like to thank Drs. Robert
Dunstan, Joana Sonea, Wasito, Elizabeth Lyons, Edmund Rosser, Edward Mather,
Schillhorn Van Veen, and Christopher Brown of the College of Veterinary
Medicine at Michigan State University for their technical support and guidance.
I would also like to thank Dr. Richard Merritt, Vlfilliam Morgan, James Kidder,
and Dan Kelly for the use of their research facilities.

I would like to express my deepest gratitude to Dr. Harold D. Newson for
his academic and emotional support and to Dr. Willis W. Wirth for his
assistance in identifying the ceratopogonids specimens collected during this
field study. I am also grateful to Drs. Robert F. Ruppel, Roland L. Fischer, and
John B. Kaneene for serving on my committee. I would like to thank Bassey Eyo,
Kathy Smith, Hastari Wuryastuti, and Anthony D' Angelo for their warmth and
friendship.

I would especially like to thank my wife, Teresa for her support and the

numerous sacrifices which she endured.

TABLECFCXJVTENTS

Page

LIST OF TABLES ................................................ v

LIST OF FIGURES ................................................ vi

INTRODUCTION .................................................. 1
LITERATURE REVIEW ............................................. 3
QuIiQQiggs Vectors ............................................ 3
Taxonomy and Biology of Qulimides W .................... 4
Taxonomy and Biology of Q. gendgaljs ............................ 8
Distribution and Prevalence of Equine Onchocerciasis .............. 13
DESCRIPTION OF THE STUDY AREA ................................. 15
MATERIALS AND METHODS ........................................ 22
Equine mum Infections ................................... 22
Natural Infection Rate ......................................... 27
Light Trap Collection .......................................... 28
Attempts to locate the breeding sites of Q. m . ............ 32
Experimental Infections ....................................... 34
RESULTS ........................................................ 43
Equine W Infections .................................... 43
Natural Infection Rate .......................................... 47
Light Trap Collection .......................................... 55
Attempts to locate the breeding sites of Q. My: .............. 55
Experimental Infections ........................................ 61
DISSCUSSION .................................................... 80
SUMMARY AND CONCLUSIONS ....................................... 88
LIST OF REFERENCES .............................................. 89

UST OF TABLES

Table Page
1. Quflggides collected from horses within 1/4 mile of
site A ..................................................... 19
2. Morphological dimensions of microfilariae from skin
biopsy .................. - ................................. 46
3. Qujjgoides collected from an infected horse at site A
in 1985 .................................................. 49
4. Quljggidgs collected from infected ponies at site B
in 1985 ................................................... 51
5. Natural filarial infections of Quflgqidgs ....................... 52
6. Blacklight trap collection of Quljmjgfis and other Ceratopogonids
at site A in 1985 .......................................... 58
7. Blacklight trap collection of Qujjmjdas and other Ceratopogonids
at the DeWitt stable in 1985 ............................... 59
8. Blacklight trap collection of Qulimidas and other Ceratopogonids
at the MSU Veterinary Research Farm in 1985 ................ 60
9. Development of W In wild caught
Q. y. yarijpgnnis ........................................... 71
10. Experimental infection of laboratory reared
Q. y. yan'jnamis ........................................... 72
11. Development of Qnghggema' II‘I laboratory reared
Q. 1. yarjjgemis ........................................... 79

USTOF FIGURES

Figure Page
1. Location of site A and B ..................................... 16
2. Study site A .............................................. 17
3. Study site B .............................................. 20
4. Microfilariae found in the midgut of an unknown black fly species

which had fed on the infected horse at Site A (x 125) .......... 23
5. Microfilariae from the above black fly seen at a higher

magnification (x 313) ..................................... 23
6. Blacklight trap used to collect Qujjggiggs .................... 29

7. Blacklight trap in operation at the Veterinary
Research Farm ........................................... 3O

8. Blacklight trap in operation near the west entrance of the stable

at site A ................................................. 31
9. Emergence traps on the manure pile in the west pasture

at site A ................................................. 33
10. Breeding site of Q. 1. yarflpgnnjs near the drain pipe outlet in

the south pasture at site A ................................. 36
11. Breeding site of Q. y. yafljpgnnis near the base of a compost pile

on the tree nursey at Michigan State University ............... 37
12. Nylon feeding bag ........................................ 39
13. Nylon feeding bag attached to the ventral abdomen of an

infected pony at site B .................................. 40
14. Nylon feeding bag attached near the umbilicus of the pony ...... 41

vi

15.

16.

17.

18.
19.
20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

vii
Microfilaria ingested by Q. am after feeding on an

thgmma infected horse (x 500) ............................ 44
Microfilaria ingested by Q. 5131mm after feeding on an infected
horse (x 500) .............................................. 44
Microfilaria ingested by Q. 1. W after feeding on the
infected horse (x 400) ...................................... 45
Microfilaria from the skin of an infected pony (x 500) ........... 45
Microfilaria in the skin of an infected pony (x 500) .............. 48
Quahggema type larvae in the thoracic muscles of wild caught
Q. m (x 313) ......................................... 53
Qnghgcema type larva (broken specimen) dissected from the
thoracic muscles of wild caught Q. 51911113: (x 200) ............. 53
Quohgcema type larva dissected from the thoracic muscles
of wild caught Q. 1. mm (x 125) ........................ 54
Qnghmgma type larva in the thoracic muscles of the above
Q. 1. yan'jpgnnjs midge (x 125) ............................... 54
Non—W type larva (arrow) in the thoracic muscles
of wild caught Q. m (x 125) ........................... 56
Non-Qngnggema type larva (arrow) in the thoracic muscles
of wild caught Q. stelliter (x 313) ............................ 56
Non-anngcema type larva (arrow) in the thoracic muscles
of wild caught Q. W (x 160) ........................... 57

Qngngcema type larva (arrow) in the thoracic muscles of Q.
QDSQLQtui 2 days after feeding on an infected

horse (x 160) ............................................... 63
Qnghgggma type larva (arrow) dissected from the thoracic

muscles of Q. Ma 11 days after feeding (x 125) ........... 63
W type infective (L3) larva in the head of Q. 1.

mom 6 days after feeding on the infected horse (x 125) . . . . 64

Same infective (L3) larva in the proboscis of Q. 1.
188112980136 313) ......................................... 64

The infective (L3) larva emerging from the proboscis (x 400) ....... 65

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

viii
A second third stage infective (L3) larva (arrow) found in the
head of the same Q. y. yadjpgnnjs (x 80) ...................... 65

The infective stage (L3) larva dissected from the head of Q. 3;.
yan'jpgnnis (x 125) ......................................... 66

Second stage filarial larvae in the thoracic muscles of Q. y.
W 7 days after feeding (x 160) ....................... 67

Second stage filarial larvae in the thoracic muscles of Q. y.
W 8 days after feeding (x 313) ...................... 67

Second stage filarial larva (arrow) in the thoracic muscles of
Q. 1. yanjggmjs 10 days after feeding (x 125) .................. 68

Second stage filarial larva (arrow) in the thoracic muscles of
Q. 1. yafiipgnnis 11 days after feeding (x 125) .................. 68

Second stage filarial larva (arrow) in the thoracic muscles of
Q. 1. yafijpgnnis 18 days after feeding (x 125) .................. 69

Second stage filarial larva (arrow) in the thoracic muscles of
Q. y. 13mm 20 days after feeding (x 44) ................... 69

lnfective (L3) filarial larva emerged from the head of Q. 1.
W 25 days after feeding (x 80) ....................... 70

Second stage filarial larva (arrow) in the thoracic muscles of
clean Q. 1. W 4 days after feeding (x 125) ............. 74

Second stage filarial larva (arrow) in the thoracic muscles of
Q. x. mm 6 days after feeding (x 125) ................... 74

Second stage filarial larva (arrow) in the thoracic muscles of
Q. 1. Magnum 14 days after feeding (x 160) .................. 75

An infective (L3) larva in the head of_Q. y. yafijpemjs 23
days after feeding (x 50) .................................... 75

lnfective (L3) larva emerging from the proboscis of Q. 1.
W (x 200) ......................................... 76

lnfective (L3) larva emerging from the mandible of Q. 1.
yafijpgnnjs (x 160) ........................................ 77

Second stage larvae in the thoracic muscles of Q. 1.
yafijnannjs 7 days after feeding (x 160) ...................... 77

ix
48. Second stage larvae (arrow) in the thoracic muscles of Q. 1.
yacilpennis 12 days after feeding (x 160) ....................

Equine onchocerciasis is a chronic infection in equines caused by filarial
nematodes of the genus annmma (Diesing). These filarial infections are
rarely recognized as a cause of ill health in equines (Nelson, 1970). However,
they have been associated with four pathological conditions: periodic opthalmia,
remittent dermatitis, poll evil (atlantal bursitis), and fistulous withers. Some
authors consider W infections as either a direct or indirect cause of
these conditions (Robson, 1918; Hall, 1923; Roberts, 1963; McMullan, 1972;
Herd and Donham, 1983). According to Cello (1971), the ocular lesions observed
in the opthalmia are nearly identical to the lesions seen in human cases of
onchocerciasis. Studies of mm infections in equines could contribute to
the possible control or elimination of the disease in man.

Previous studies of W infections have been concerned with
prevalence of the microfilariae in the equine host (McCullough et al., 1977;
Stannard and Cello, 1975), the morphology of the parasites (Gibson, 1952;
Mellor, 1974a) and transmission of the gamma spp. under laboratory
conditions (Mellor, 1971; Collins and Jones, 1978). There have been few studies
on the natural transmission of W to equines. In 1933, Steward

incriminated Quljggjm (Diptera: Ceratopogonidae) as vectors of equine
W in England. He concluded that Q. W (Meigen) was a natural

2
vector of Q. mus (Railliet and Henry) and Q. Mus and Q. pagan
(Kieffer) also were probable vectors. Since then there have been few studies on
the natural transmission of Onchocama infections in equines. According to
Wirth (1977), this may be due to their small size and difficulty of colonization.
However, some authors have attempted to identify potential vectors in the field.
In Australia, Ottley and Moorehouse (1983) collected engorged wild

ceratopogonids and black flies (Diptera: Simuliidae) from infected horses in the

field- They reported that W (Lasiobalaa) WIS. (Taylor)
(Diptera: Ceratopogonidae) and a black fly, W pesjflgns (Mackerras

and Mackerras) may be potential vectors. Foil et al. (1984) collected engorged
Qujjggiggs from a pony baited stable trap in Louisiana. Filarial nematodes were
found in the thoracic muscles of only Q. m (Coquillett) and they
concluded that partial development of Q. gandgafls occurs in this species.
Recently, equine onchocerciasis was reported from horses in Michigan, but
no mention was made of the vector (Schmidt et al., 1982). Two suspected

vectors, Q. 91252191115 and Q. variipennjs yarijpennjs have been reported from the
state. The purpose of this study was to evaluate the role of Qujjggmfi,

particularly Q. gusgjejus as potential vectors of W in Michigan equines.

LITERATURE REVIEW

E l' . l I! |

Two species of Qufiggiges, Q. W and Q. yaflipennjs have been
demonstrated to be vectors of W candgafis under laboratory conditions
(Mellor, 1971 ). He used a membrane feeding technique to infect both species of
Qujjggjges with Q. Wis microfilariae extracted from the skin of an
infected horse. lnfective (third stage) larvae developed in 5.5% of 232 Q.
W and 7.7% of 117 Q. WW (Coquillett) females.
Mellor (1975) fed laboratory bred Q. W and Q. xariinennis mm
on the abdomen of a horse with 7 mff/mg of skin. He found 28 of 169 (16.6%) Q
W and 24 of 329 (7.3%) Q. yafljpgnms W had ingested Q.
mus microfilariae, thus demonstrating that these two species will feed
on infected animals under field conditions. In other studies both midges have
been observed to feed on horses under field conditions. (Steward, 1933; Jones et
aL,1977)

In England, Steward (1933) reported a 5-6% natural infection rate in field
collected Q, W and he also observed the development of Q migaljs
to the infective stage in these wild flies. In the United States, Foil et al. (1984)
collected engorged Q. W females from a pony baited stable trap in
Louisiana. They found 8 of 114 (7%) Q. W infected; one third stage

4
larvae was observed in a single midge. According to Moignoux (1952), Q
uaaegulgsgg is a vector of Q Lgflggmtg (Diesing) of horses in Europe. He
recorded a 2.1% natural infection rate and a 29% experimental infection rate.
Supperer (1953) suggested that this parasite was not thjgulata but Q.
. l' .

Quflgajggg uybegujggug and Q. milagunm are closely related
taxonomically; they belong to the same subgenus Monoculicoides. Qujjggidgg
ugaggujggug is distributed in Europe and Asia and Q yanjaguum occurs in North
America from Canada to Mexico. Qujjagiagg yaniaguum is considered to be a
species complex in North America, consisting of 5 subspecies. According to
Jamnback (1965), the subspecies found in Michigan is Q ganjagunm ygnjaguum.

Other Qaljggiags spp. have been reported as possible vectors of nguaggrca
species in equines. According to Steward (1933), Qgtmglgtu; and Qagrrgti
may be vectors of Qggungalm. The midges were found to be infected with
microfilariae after feeding on an infected horse. He reported that 9 of 172
(5.2%) Q. gagglgtug and 1 of 6 Q. agngti ingested microfilariae. Apparent
development of microfilariae was noted in one Q. gbsglgtug which had a blood
meal 3-4 days earlier.

I IE' I [C l' 'l | l I

The Q. gagglgtug group has a worldwide distribution, the species group is
found in Eurasia, North Africa, and North America. In the United States it is
comprised of 4 species: Quliggidgs glaguug (Jamnback and Wirth), Q. atmalgnm,
Qgguguiguga (Coquillett), and quigatgum (Meigen). Quliggidgg glggnag is

5
found in northern Florida and South Carolina (Blanton and Wirth, 1979), while
the other three species have been reported from Michigan (Jamnback and Wirth,
1963; Williams, 1955). Jamnback and Wirth (1963) and Jamnback (1965) listed
the characters used to separate species in the 2mm group in the United
States.

Only the female Quliggiagg bite. A blood meal is required for the
development of the eggs and digestion of the blood meal is completed in 4-5
days at 70°F (Jamnback, 1961). He also noted that the first egg batch may be
autogenous. According to Hill (1947), the female midges may take as many as
5 blood meals before laying their eggs. Jamnback (1961) reported that 9 of 17
wild collected Q gagglgtgg females laid eggs 6-7 days after a single blood
meal; the eggs hatched 4-7 days later. Service (1969) noted that the eggs
hatched in 3 days. The larvae pass through 4 molts (Lawson, 1951) over a 3 1/2
- 5 months period (Hill, 1947). Jamnback (1965) suggested that Q. gagajgtug may
_ overwinter in the larval stage. Pupation occurs in the dry portion of the breeding
medium (Hill, 1947).

The immatures have been collected from a wide range of habitats. Hill
(1947); Kettle and Lawson (1952) found Q gbsglgtug breeding in sphagnum bogs
and marshes. Murray (1957) collected the midges along sandy stream banks, in
tree holes, and piles of damp leaves. The temperature of the latter breeding
site averaged 71°F. Jones (1961) reported Qgtmglgnm breeding in a pile of
cow manure and bedding, and in decaying cornstalks. Both breeding habitats

were exposed to direct sunlight and their surfaces were dry. Hair et al. (1966)

6

collected the midge from a pile of chicken litter in an open field. The majority
of the larvae were found several inches below the dry surface in the moist
layer. The midge has been found in alkaline water habitats (Kardatzke and
Rowley, 1971), moist straw, decaying spruce leaves (Jamnback,1965), and
decomposing banana stems (Birley et al., 1984). Most Quflagjagg pupae of other
species can be collected by flooding the breeding media with water, however,
the ggsngnm group pupae remain submerged (Jamnback, 1965). Adult Q.
gagglgtyg emerge about 5 days after pupation (Hill, 1947; Jamnback, 1965).
Jamnback (1961) reported that wild Q. gtmajgtug females lived for 51 days
under laboratory conditions.

Little is known of the flight range of adult Q.ga_gg1g1u§_. It has been
suggested that Qujjgnggg do not usually occur far from their breeding sites,
but that the wind may disperse them beyond their normal flight range (Hill,
1947). Birley et al., (1984) collected Q._gb_sg|g1ug about 400 m from a known
breeding site, a banana plantation. The prevailing wind was blowing in a
westerly direction across the plantation towards the collection site.
Shemanchuk (1972) concluded that the wind acts as a passive mode of transport,
since wind velocities greater than 10 mph hindered flight of the midge.
Jamnback and Watthews (1963) observed that an average wind velocity of 1.5
mph reduced the landing rate of gagglgtug group females to zero. The flight
activity of Q. ghsglgjgg also is influenced by other climatic conditions.
Jamnback and Watthews (1963) recorded large numbers of the $32.50.]ng group

females landing on a human host at temperatures between 55 and 81° F, and

7
noted that the midges were inactive at temperatures below 50° F. Shemanchuk
(1972) recorded Q. gagglgtag activity from 46 to 72 ° F with optimum activity
occurring between 54 and 63° F. The midges were also active during a light
rain.

The adults of Q. gtmglgtag occur from early spring to late autumn.
Jamnback (1965) collected them from May-September in New York and
Jorgensen (1969) observed them in southeastern Washington until late October.
Hill (1947) reported Q. gagglgjug from early April to late October in England; the
population peaked in early June and again in late September. From this she
suggested that Q. gagglgnm has two generations a year. Murray (1957) found the
midges from June to September in Virginia and reported that the population
peaked twice, from mid June to mid July and from late July to mid August.
Service (1969) found the midges until November in southern England and
recorded only a single population peak in May. Schmidtmann et al. (1980)
considers Q. gtmglgtug to be multivoltine in central New York. In Michigan,
Williams (1955) collected the midge from late June to early August; they were
most abundant in July.

The adult midges have been reported to be active throughtout the day.
According to Hill (1947), Q. gtmgIgnm is most abundant in the early evening
hours, beginning 3 1/2 hours before sunset. Others have observed midge activity
during the daylight hours in shaded areas or on cloudy days (Service, 1969;
Twinn, 1931), and in wooded areas (Williams, 1951). Murray (1957) reported

large numbers active between 2 and 5 am. in the morning. Williams (1955)

8
suggested that light intensity has a major influence on the activity of Q.
9.1321291113-

Qujjggiagg gagglgtug has a wide host range, it feeds on horses, cattle,
sheep, man (Twinn, 1931; Jamnback, 1965; Schmidtmann et al.,1980) and birds
(Bennett, 1960). Schmidtmann et al. (1980) collected Q. gtmglgnm from the
belly, back, brisket, and neck of ponies in New York but the majority of the
females were found on the belly. Mellor (1974c) reported that the gagglgtug
group females occur on all parts of the horse, but they were most numerous
along the ventral abdominal midline. They have been reported to attack
vertebrate hosts located from 0-25 ft above the ground (Bennett, 1960; Service,
1969). According to Service (1969), the majority of Q. gagglgtug occur below 10
ft from the ground.

I IE' I I Q . I.

In 1910, Railliet and Henry reviewed the genus ngugagrga and separated
ngugggmgagmigglm from the type species ngugggma rgtjgulatg. The
taxonomy of Qggnngalm has caused much controversy; some authors still
consider Q ggungglm as a synonym of Q. Lgtjgulgtg. According to Sandground
(1934), the only morphological difference between the two species is the length
of the left spicule in the adult male. Others consider the two parasites as
separate species. Le Roux (1950) reported that the "rugae" are more closely
spaced in Q rgtigulatg than in Q. ggungglm. He also thought Q. ggungalm might
be a probable synonym of Q. gamma (Neumann). Bain (1975) re-examined both

parasites and suggested that distinct morphological differences exist in the

9
adult female cuticle. Lichtenfels (1975) used several characters to
differentiate between the two species: the anatomical location of the adults in
the host; length of the left spicule in the male; and the morphology of the
female cuticle. The site of development of the adult parasites has been
commonly used by authors to distinguish between the two species.

The taxonomy of the immature stages of nguaagrga spp. in equines is as
controversial as the adult stage. Some authors have separated the two species
based on their differences in size (Supperer,1953; Lichtenfels,1975). According
to Gibson (1952) and Mellor (1974a), the size of the microfilariae is of little
taxonomic importance unless the fixation and staining method is indicated,
since the size of the microfilariae is affected by these procedures. The location
of fixed internal morphological structures: the first nucleus; anterior edge of
the nerve ring; excretory pore; excretory cell; rectal cell; anal pore; last
nucleus; and length of the tail from the anterior end of the microfilariae has
been used to distinquish between ngugggma spp. (Steward, 1933; Gibson, 1952;
Collins, 1973; and Mellor, 1974a). Moignoux (1952) reported that the
microfilariae of both Q. gguugalm and Q. fgtjgulgtg develop in Q. uguggglggug
and the larvae are morphologically similar. According to Gibson (1952), the
morphology and arrangement of the caudal nuclei can be used to identify Q.
Lgtjgujgtg (= agnnggjjg), but Mellor (1974a) did not observe these same
characteristics in the caudal nuclei of Q. ggungglm microfilariae extracted
from the skin of infected horses in England.

The adults of Q. ggungalm are whitish, thread-like nematodes. The cuticle

10

is transversely striated with external spiral thickenings, which are intermpted
in the lateral field; there are 3 to 4 transverse striations between each ring
(Railliet and Henry, 19l0; Lichtenfels, 1975; Bain, 1975). The adult female
parasites are 35 to 38 cm long and 0.2 to 0.35 mm wide, and males are 6 to 7 cm
long and 0.07 to 0.10 mm wide (Steward, 1933). The viviparous females produce
large numbers of unsheathed microfilariae which range in size from 160 to 240
u long and 2 to 5 u wide (Steward, 1933; Levine, 1968). In microfilariae stained
with nuclear stains, dense staining bodies or nuclei are arranged in a column
formation, 1 to 3 abreast (Mellor, 1974a). The adults of Q. ggungalm are usually
found in the ligamentum nuchae of the equine (Mellor, 1973a). This large neck
ligament consists of a funicular and Iamellar portion (M' Fadyean, 1902). The
parasite is found predominately in the funicular portion between the last
cervical and the fourth thoracic vertebrae (Mellor, 1973a). Ottley et al. (1983)
found adult Q. ggrnggjm in both the ligamentum nuchae and the scapular
cartilage of a horse in Australia. The microfilariae are found mainly in the skin,
at a depth of 1.6 to 1.8 mm from the surface; no periodicity of the microfilariae
occurs in the skin (Steward, 1933). The microfilariae are rarely found in the
blood, however, Thomas (1963) reported finding an unknown ngugcgrag species
in the blood of equines in South Africa.

According to Mellor (1974a), two sizes of Q. ggungalm microfilariae occurs
in the horse: a uterine, large size and a skin, small size. He suggested that the
skin microfilaria is the mature form. Mellor (1973a) reported that the

microfilariae are distributed unevenly in the skin and throughout the body of the

11

horse; they congregated in isolated clusters in the skin instead of being evenly
distributed. Rabalis and Votava (1974) found large numbers of microfilariae
along the flank and the umbilicus proper of the horse. Collins (1973) reported
finding the highest concentration of microfilariae in the skin near the withers
and the ventral region posten'or to the forelegs of the horse. Mellor (1973a)
recorded 95% of the microfilariae from the skin along the ventral abdominal
midline.

The method of distribution of the microfilariae in the skin of equines is
poorly understood. It is thought that the microfilariae migrate through the skin
to various areas of the definitive host. Mellor (1974b) suggested that the
microfilariae are guided by a stimulus, such as a chemical gradient or a
combination of co-evolutionary factors between parasite and vector. El
Sammani and Hussein (1983) suggested that the microfilariae of Q. Lailljgtj
(Bain, Muller, Khamis, Guilhon, and Schillhorn Van Veen) may migrate to the
preferred biting site of the vector.

The longevity of Q. ggmjggjm microfilariae in the skin is unknown,
however, the microfilariae of ngugggrgaygjyujug (Leuckardt) may survive in
human skin for 1-3 years (WHO, 1966).

ngugggugaggnugaflg microfilariae continue their development in an
intermediate host, leiggiggg spp. (Steward, 1933). The midge ingests the
microfilariae from the skin of the equine, along with blood from ruptured
capillaries. According to Mellor (1975), the microfilariae are either ingested

from the area near the damaged skin tissue or they are attracted to the vector

12
by a chemical attractant in the midge's saliva. The ingested microfilariae and
blood meal pass to the midgut of the midge. The microfilariae can be found in
the midgut 1-3 days after a blood meal (Steward,1933; Mellor, 1975). However,
Mellor (1975) found Q. agmigalm microfilariae in the haemocoel and thoracic
muscles of Q. ygnjaguujg and Q. nuaggulagug approximately 5 and 15 minutes,
respectively, after blood feeding. He suggested that the microfilariae enter the
haemocoel by penetrating through the midgut wall. Development of the
microfilariae continues in the thoracic muscles but at an unequal rate (Steward,
1933). After 7 days, the microfilariae have reached the ”sausage form” or 2nd
stage larvae; they are 133-240 u long by 17-27 u wide. By the 16th day, they
begin to increase in length and decrease in width. The infective larvae (third

stage) may be found in the head of the midge on the 22nd day and in the
proboscis 24-25 days after an infected blood meal. The infective (L3) larvae

measure 600-700 u long by 18-21 u wide (Steward, 1933). The infective larvae
enter the equine host when the midge feeds again. The parasite may emerge from
the membranous area at the base of the proboscis during the feeding process
(Steward, 1933).

Temperature has been shown to influence the rate of development of Q.
agunggjm in the Quljggjggg vector. Steward (1933) made no mention of the

temperature at which he observed the development of Q. smungalm to infective
(L3) larvae in Q. ungggulgsag. Mellor (1975) observed the complete development

of Q. ggunggjmm Q. ngbggujggagand Q.yg[j_iaguumin 14to 15 days at 69.8 to

13
73.4°F, but he did not indicate the anatomical location of the infective (L3)

larvae in these flies. lnfective stages were not observed in the heads of the

midges. Collins and Jones (1978) reported that Q. cgungalm developed to the
infective (L3) stage in Q. yarjjagnnjg in 5 days at 80°F and 12 days at 71°F and

were distributed in the head, thorax, and abdomen of the flies. Moignoux (1952)
observed the complete development of Q. rgtigulata in Q. nuhggulgsugin 22 days
at 77-86°F. He mentioned that the parasite was also able to complete

development at 60.8°F, but did not state the developmental time at this lower
temperature. The activity of the L3 larvae in the equine host is unknown. It is

assumed that they migrate to the ligamentum nuchae and there continue their
development to the adult stage (Mellor, 1974b). The length of time from
innoculation of the infective stage until the microfilariae occur in the skin has
not been determined for Q. ggnrigglig but for Q. yglyulug in man it takes 4-5
months (McMullan, 1972).
EH“ IE I [E' El ..

ngugagrga spp. infections in equines have a worldwide distribution. They
have been reported from Europe, Asia, North America, Central and South
America, Australia, Africa, and the Middle East (Bain et al., 1976; Ottley et al.,
1985). In the United States, ngugggrga infections in equines were first
observed by Van Volkenberg in 1921. He also found parts of another nematode
parasite in the ligamentum nuchae, but without the spiral thickenings seen on

the cuticle of Qngugggma. According to Lichtenfels (1975), Q. agungalm may be

14
the only anhccarca sp. occurring in North American equines. Two methods have
been used to diagnose nguaagmg infection in equines: demonstration of the
adult parasite in the ligamentum nuchae; and/or the presence of the
microfilariae in the skin (Levine, 1968). Lloyd and Soulsby (1978) found 74 of
121 (61%) equines from the eastern United States infected with Q. ggungalm
microfilariae. Mc Cullough et al. (1977) reported that 29 of 57 (50.8%) horses
biopsied in Maryland were positive for Q. ggudgaljg microfilariae. Cummings
and James (1985) found 341 of 664 (51.4%) horses from the southeastern and
midwestern United States with cutaneous Q. ggmigalm microfilariae. Stannard
and Cello (1975) found 48 of 100 (48%) horses from California, Oregon, Nevada,
and Arizona infected with Q. gguuggjm microfilariae. Microfilariae were
extracted from tissue samples collected from the eye, ventral abdominal
midline. and the lower eye lids of the infected horses. Polley (1984) collected
skin biopsies from areas adjacent to the umbilicus of 623 horses from western
Canada and the northwestern United States and reported a microfilarial
prevalence rate of 25.8%. Collins (1973) found Q. agndgalm microfilariae in 52
of 232 (22%) horses in southeastern Louisiana. In Michigan, Schmidt et al.
(1982) collected a section of the ligamentum nuchae from 83 horses and
reported finding adult Q. ggmjgal‘m in 31 (37%) of the ligament sections

examined.

DESCRPTDNCFTHE STLDYAREA

The study was conducted at two sites in Clinton County, Michigan (Figure
1). Site A is a 19 acre farm located 1/4 mile north and 2 miles west of the
Capitol City Airport (Figures 1, 2). A case of equine onchocerciasis had been
reported from this farm in 1977. The farm consists of a house, garage, storage
shed, stable, pasture, and a maple-beech woodlot. The owners of the farm are
horse breeders: they usually have 7-10 horses on their property throughout the
year. There are also 5 chickens, 5 cats, 3 nesting pairs of barn swallows, 2
peacocks, and 2 dogs on the farm. Except for a stallion, the horses are rarely
kept in the stable and are allowed to roam on the pasture day and night.
The pasture is divided into 3 sections: a north, south, and west section.
The west pasture is the largest, covered by an abundance of low growing scrub
vegetation and horse manure. The contour of the pasture is irregular. It slopes
5-20 degrees along a 634 ft by 528 ft perimeter, then levels off near the
entrance of the woodlot. The temperature is usually several degrees cooler and
the wind is not as strong in this depression as at the surface of the ridge. The
maple-beech woodlot covers 6.3 acres and contains two temporary ponds
located in the woodlot. The north and south pastures are grass covered
savanna-like, except for a single apple and black walnut tree in the north and
south pastures, respectively. The horse bedding and manure removed from the

horse stalls is dumped in several large piles in the south and west pastures. A

15

 

GRAND RIVER

 

 

 

 

 

STATE ROAD

 

 

 

 

STULL ROAD

 

 

Figure 1. Location of site A and B.

DUCK
POND

    

MAINTENANCE
COMPANY

 

GOAT FARM
WATSON AND SUMMERS
DRAIN
S
E W
N

Figure 2. Study site A.

18
drain pipe from the farm house extends into the south pasture and its effluent is
discharged on the ground surface, which also contains a mixture of horse
manure, maple leaves, black walnut leaves and nuts. The sewage effluent flows
for 20-30 It into the pasture. The surface of the soil has a sandy loam
consistency and a black foul smelling bottom layer. This area remains moist
throughout the year because of the daily discharge of water from the drain pipe.

A tree nursery is adjacent to the western edge of site A. A 211 ft by 370
it natural pond is located on this property and the Watson and Summers drain
flows along the back of the nursery. The pond and the drains are located about
422 ft and 1162 ft respectively from the woodlot. The site (Figure 2) is
bordered on the north by a corn field and a goat farm, on the south by a house
and maintenance service company, and a dirt road on the east.

A preliminary collection of wild Qaflagiagg landing on the ventral abdomen
of horses at and within 1/4 mile of site A, showed that Q. ggsgIgtug occurred in
the area (Table 1).

Site B (Figure 3) is a 2 1/2 acre residence located 1 mile east of site A. It
consists of a house, storage shed, garden plot, stable, and a pasture. Both the
pasture and stable are covered with a thick mat of pony manure. The owners
have 5 ponies which are mainly used for pleasure; all of the ponies were foaled
in Michigan. The ponies usually spend most of the time along the west edge of
the pasture or inside the stable on the north side of the pasture. Other animals
on the property are a cat, a goat, 2 nesting pairs of barn swallows and 3 dogs.
The pasture is mostly flat ground with a sparse growth of vegetation and scrub;

it is bordered on the west by the owner's house, a storage shed, and a garden

19

Table 1. Quljggiggs collected from horses within 1/4 mile of site A.*

 

 

 

Species Site A Fisher's Farm Subtotal
Q. QbfiQISIIIS 55 35 90
Q. gtglljfgr 189 759 948
Q. It. Mafllnennls O 3 3
Total 244 797 1 041

 

* Collected from July 17 to October 12 (1984).

20

  

MARSH AREA

cf

4' uni-(-
1.37331???

 

 

 

STATE ROAD

 

HOUSE

 

Figure 3. Studg site B.

21
plot. A drainage ditch extends from the garden plot along the entire length of the
north side of the pasture. The pasture is bordered on the north by an open field,
on the east by a field covered with a dense growth of vegetation and scrub, and
the south by a dirt road. Across the dirt road is a corn field with a marshy area

at the far side.

MATERIALSANDNEn-KDS

E . Q I l l I.

A horse from site A had been diagnosed positive for onchocerciasis on
August 30, 1977 by the veterinary medicine clinic at Michigan State University.
However, the owner had no record of which horse was examined that day. She
assumed it may have been one of the older horses. According to the owners at
site B, their ponies had never been examined for onchocerciasis. The equines
from both sites were examined at the beginning of this study by a veterinarian,
Dr. E. J. Rosser from Michigan State University. According to Dr. Rosser, clinical
signs of onchocerciasis were noted in 2 of 3 horses at site A and 3 of 5 ponies
at site B. He suggested that skin biopsies should be taken from the skin lesions
to confirm the presence of Qngugggmg microfilaria in the tissue.

Two methods were used to diagnose ngugcgma infections in the equines:
skin biopsies and the presence of ngugggrga microfilariae in blood smears from
engorged Quliagiagg that recently had fed on one of the horses or ponies. At site
A the owner would not permit a skin biopsy to be taken from the horse called
Lacey, one of the animals Dr. Rosser thought was

infected. It had been observed from previous collections of engorged biting
flies from this horse that an unidentified black fly species ingested large
numbers of microfilariae while feeding (Figures 4 and 5) on this horse, so it

was assumed to be infected with Qngugagrga. It was a 20 year old mare, that
22

 

Figure 4. Microfilariae found in the midgut of an unknown black fly
species which had fed on the infected horse at Site A (x 125).

 

Ir-

Figure 5. Microfilariae from the above black fly seen at a higher
magnification (x 313).

24
was born and raised in Michigan. Quljggiagg feeding on it were collected from
the ventral abdomen of the horse with an aspirator beginning 1-2 hours before
and until 1 hour after sunset. Initially, a battery operated aspirator was used to
collect the midges, but had to be abandoned because it frightened the horse. A
tube aspirator described by Endris et al. (1982) was tried, but was discontinued
because the collector became allergic to inhaled foreign particles from either
the horse or the midges. An aspirator described by Hill (1947) with slight
modifications was used. The glass tubes inserted in the cork were replaced with
plastic tubes and 10 dram clear plastic collection vials were filled to a depth of
1 inch with plaster of paris and allowed to dry. Before field use, 2 inch by 2 inch
pieces of saran cloth were placed on top of the vials, then covered with plastic
lids with the center portions removed. The construction of these vials is similar
to the cage used by Endris et al. (1982) for rearing sand flies (Diptera:
Psychodidae) in the laboratory. The vials were placed inside a 2 qt square
polystyrene food container, the bottom of the container was lined with damp
paper towels and the plastic lid was placed over the container. The collection

vials were transported to and from the field in this manner.
In the laboratory, the midges were anesthetized with 002 from dry ice or

chilled in the freezer compartment of a refigerator for 45-60 seconds. They
then were sorted in a glass petri dish into engorged and unengorged groups using
a dissection microscope (magnification 40x). The unengorged flies were killed
with an alcohol saturated camel hair brush and stored in 70% ethanol. The
engorged flies were placed in 3 ml of 0.9% saline in a 5 ml blood tube. The blood

tube was sealed with a rubber stopper and the solution was shaken vigorously

25
until the flies settled to the bottom of the tube. They were left in the saline
solution for 20-30 minutes, then dissected in a drop of saline solution on a
microscope slide under a dissection microscope. Minuten insect pins embedded
in wooden applicator sticks were used to separate the abdomens from the thorax
and head segments (the latter two segments were placed in 70% ethanol in 5 ml
blood tubes until their species identification was confirmed). The abdomens on
the slides were gently teased apart to remove the blood mass, additional saline
was added to completely dissolve the blood mass and then the preparations were
allowed to dry overnight. The dried blood was stained either with Giemsa, using
a method described by Belding (1942), or Mayer's Acid Hematoxylin (1 part stock
stain/ 9 parts distilled water by volume) using a method similar to Menon
(1960) except the slides were maintained in the stain for 10 minutes and rinsed
in distilled water (ph 7.0) for 1 minute.

The second method routinely used to diagnose nguaagnga infection in
equines was conducted at site 8. Two ponies were biopsied, one was a 2 year old
mare named Pepper, the other a 14 year old stallion named Comanche. A single
biopsy sample was obtained from the abdominal midline of Comanche and from
an abdominal lesion of Pepper. Each pony was administered 3 ml of Xylzin
(Rompun) intravenously and the biopsy sites were scrubbed with a 2 inch by 2
inch sterile gauze saturated with a betadine/soap solution. The hair was
removed from the biopsy site with a disposable razor, then the area was
scrubbed again with betadine and allowed to dry. The site was infiltrated with 3
ml of lidocaine and 6 mm Baker's biopsy punches were used to remove the skin

samples. Each sample was cut in half with scissors. One piece was mounted on a

26

wooden tongue depressor and placed in 100 ml of 10% buffered formalin in a
plastic virology container and submitted to the Animal Health Diagnostic
Laboratory at Michigan State University for histopathological examination. The
other piece was placed in 100 ml of 20% Tyrode/Horse Serum solution (THS) in a
plastic virology container. Slight bleeding was observed after removing the
biopsy specimen so a 2 inch by 2 inch sterile gauze pad with digital pressure
was used to control the bleeding. A tetanus antitoxin was administered
subcutaneously to both ponies. The skin samples were transported back to the
laboratory approximately 2 hours after removal.

In the laboratory, the skin samples were removed from the THS solution
with sterile forceps, rinsed in fresh THS solution, cut into small strips, using
sterile scissors, and placed in a 6 ml plastic culture tube. Two ml of THS, 200
ug of streptomycin, 200 units of penicillin, and 100 units of nystatin were
added to both culture tubes. The two culture tubes were held in an incubator at
37°C for 18 hours to allow microfilariae to leave the tissue. The supernatant
from each culture tube was poured into a clean culture tube, the incubation tube
was rinsed twice with 2 ml of Tyrode solution, then the solution was poured
into the clean tube. Both tubes were centrifuged at 1500 rpm for 15 minutes,
the supernatant decanted, then 1 ml of 2% neutral buffered formalin (2 ml
buffered formalin/ 98 ml Tyrode solution) was added to the residue in each tube.
The tubes were centrifuged again at 1500 rpm for 15 minutes, the supernatant
decanted, then the residue was removed with a pasteur pipet and smeared on a
microscope slide and allowed to dry over night. The slide smears were immersed

in 1% glacial acetic acid, then 1% sodium bicarbonate, rinsed in distilled water,

27
then 4% aluminum ammonium sulfate, rinsed in distilled water, then placed in
Mayer's Acid Hematoxylin (1 part stock stain/9 parts distilled water) for 5
minutes in each solution, then rinsed in distilled water. After staining, the
smears were dehydrated in 35%, 50%, 70%, 80%, and 95% ethanol (1 minute in
each solution), then immersed in Euparal essence for the same amount of time.
The smears were mounted in euparal vert under a No. 2 cover glass and held in an
incubator at 55°C for 3-4 days.
NaturalJnteflimfiate

Wild adult Quligajdgg were collected by aspirator, as described above for
the engorged flies, as they landed on the ventral abdomen of the infected
equines. Collections were made one evening a week at site A from May 3 until
October 25 (1985) and twice a week at site B from August 20 until October 4,
except on windy or rainy evenings. Flies were fixed in Carnoy solution for 16-18
hours at room temperature, then rinsed in 70% ethanol for the same time period,
and stored in 5% glycerine alcohol until staining.

The midges were stained in groups of 20 to 300 in 5 ml blood tubes using a
method described by Nelson (1958), except that they were rinsed in 70%, 50%,
and 35% ethanol (30 minutes in each alcohol dilution), then were placed in
Mayer's Acid Hematoxylin (1 part stock stain/1 part 4% glacial acetic acid) for
approximately 3 hours. They were rinsed in distilled water or 1% glacial acetic
acid for about 1 minute to remove excess stain, then dehydrated in 35% and 50%
ethanol ( 30 minutes in each solution), then held in 70% ethanol for 24 hours.
This method stained the filarial larvae inside the midges and allowed the

parasites to be seen during dissection.

28

The weekly collection of Quliaaidgs from each site were pooled together.
Eighty per cent of the midges collected each month were dissected and the
number of infected midges recorded. Each was dissected in a drop of glycerol on
a microscope slide under a dissection microscope (45x). The head, thorax, and
abdomen were separated and examined for filarial larvae. The larvae were
mounted in glycerol under a coverslip, ringed with euparal, and measured with a
micrometer on a phase contrast microscope.

I . ! I I E II I.

A light trap similar to the Monks Wood light trap described by Service
(1976) was used to collect Quljggidgs from May 24 until September 4, 1985. An
ElliscoR light trap with a 15 watt blacklight tube provided the light source, a
CDC minature light trap (without a bulb) with an attached collection bag was
secured to the body of each trap by a metal wire (Figure 6). The collection bag
consisted of a 15 inch long saran cloth bag with an elastic garter sewn at one
end. The other end was sewn to the threaded lid of a 4 oz urine specimen cup. A
1 3/4 inch hole was cut in the center of the lid which was screwed on to the
specimen cup.

Two of these light traps were operated one evening a week during the
collection period in 1985, one at the Veterinary Research Farm on Michigan
State University ( Figure 7) and the other was alternated between two horse
stables in Clinton County: one located at a farm on DeWitt road, about 11 miles
north of site B, and the other at site A (Figure 8).

The midges were collected in 40 ml of Carnoy solution placed in the urine

specimen cups, held in the solution for approximately 16-18 hours, then rinsed

29

 

Figure 6. Blacklight trap used to collect
E I. . I _

30

 

Figure 7. Blacklight trap in operation at the Veterinary
Research Farm.

31

 

Figure 8. Blacklight trap in operation near the west entrance
of the stable at site A.

32
in 40 ml of 70% ethanol overnight. The Quliagiagg spp. were separated from the
other specimens under a dissection microscope and stored in 5% glycerin
alcohol. Microscope slide mounts were prepared for identification for a
representive sample of each species of Quliagiags using the method of Wirth and
Marston (1968). The morphological characters listed by Blanton and Wirth
(1979) were used to identify most of the Qquagjggg and the characters
described by Jamnback and Wirth (1963) and Jamnback (1965) were used to
separate the gagglgjgg group. Identified Qujjggiggs were submitted to Dr. W. W.
Wirth, Systematic Entomology Laboratory, Gainesville, Florida for confirmation
of their identification.
EIIIIIIIII I. 'I [Clll

Two methods were used to locate the breeding site of Q. gagglgtag:
emergence traps and a flotation technique. The emergence traps were 6 1/2 inch
planting pots with 6 dram glass vials secured in the pot drain holes. The vials
interiors were coated with a mixture of lubricant grease and 10W40 motor oil.
Twenty of these traps were used in the west pasture at site A (Figure 9). Eight
were placed on manure piles and 12 on horse manure droppings in the pasture
and woodlot.

In the second atmajgnm collecting method, suspected breeding materials
were collected from within a one mile radius of the equine baited and light trap
collection sites. A six inch garden spade was used to collect soil along the base
of manure piles, soil contaminated with animal excrement, and damp soil along
the corners of horse stalls. Wet soil from the margins of drains, ponds, and

streams, and damp leaves from tree holes and wooded areas were also collected.

33

 

Figure 9. Emergence traps on the manure pile in the west
pasture at site A.

34
Soil samples were collected from the first one inch of top soil and placed in 12
oz insulated drinking cups, 2-3 scoops in each container. The foliage samples
were placed in 10 inch by 13 1/2 inch plastic ziplock bags. Since such a large
number of habitats were surveyed, no attempt was made to standardize the
number of samples collected. In addition, some pupae were collected with an eye
dropper from tree holes and horse water troughs. In the laboratory, the samples
were placed in white enamel pans and flooded with distilled water. An eye
dropper was used to collect the Quliggjdgg pupae that rose to the surface of the
water. Pupae were placed individually in 2 dram glass vials on wet cotton pads,
the vials were plugged with cotton, then maintained at 69.8-71.6°F in a dark
incubator. After the adults emerged, they were killed with an alcohol saturated
camel hair brush; the adults and pupal skins stored in 5% glycerin alcohol
overnight, then mounted on microscope slides, identified, and forwarded to Dr.
W. W. WIrth for confirmation.
E . I I l I I.

Two studies were conducted to determine if Q. gguugglis develops to the
infective stage in Q. glmglgnm and Q. 1. yafijaguum after feeding on an infected
herse in the field. In study 1, engorged wild g. gtmglgnm and Q.1.1anjaguum
were collected (as previously described) from the ventral abdomen of the
infected horse at site A. Qaljggiagsy. ygnjaguuig was easily recognized by its
larger size and its feeding mainly on or near the umbilicus. The engorged midges
were transported back to the laboratory and released into a 6 3/4 inch long by 6
1/2 inch wide by 6 inch high plexiglass cage. Except for Q1. lamaguum, no

attempt was made to separate the midges by species. The individual midges

35

were aspirated into 10 dram plastic vials with plaster of paris bottoms (as
described above). Cotton pads soaked in 10% dextrose solution were placed on
the saran cloth as a nutrient source. The vials were then placed in 2 qt plastic
food containers, lined with damp paper towel to maintain high humidity and the
lids were placed over the containers. The midges were maintained in a dark
incubator at 62.6 - 66.2°F. Each day the sugar pads were changed and dead flies
were removed, identified, then fixed in Carnoy solution overnight. The following
morning, they were rinsed in 70% ethanol for 18 hours, then stored in 5%
glycerin alcohol.

In study 2, Q. 1. yaniaguum pupae were collected from the wet soil near the
drain pipe outlet in the south pasture at site A (Figure 10) and about 8 to 10 ft
from the base of a compost pile on the Michigan State University tree nursery
(Figure 11), then placed in 4 oz urine specimen containers on wet cotton. A piece
of saran cloth was placed on top of each container, then capped with the lid (a
hole in the center), sugar pads were placed on the saran cloth and the cups were
placed in a plastic food container. The pupae were held in a dark incubator at
62.6-66.2°F until the adults emerged. Adults were maintained under these
conditions for 3—5 days before attempting to feed them on the infected equines.
In the feeding experiments the midges from the laboratory were released inside
a small plexiglass cage and removed from the cage with a tube aspirator. The
mouth of the aspirator was plugged with cotton and the midges were
immobilized by chilling in the freezer compartment of a refigerator for 45-60
seconds. Then they were gently blown into a nylon feeding bag and the neck of

the bag was closed with a metal hair clip. Approximately 25 midges were placed

36

 

Figure 10. Breeding site of Q. 1. ya_r_ijagu_u'm near the drain pipe
outlet in the south pasture at site A.

37

 

 

Figure 11. Breeding site of Q. 1. yan'iaguum near the base of a
compost pile on the tree nursey at Michigan State University.

38
in the bag.

The bag (Figure 12) consisted of 3 inch long pieces of nylon, cut from the
toe portion of a pair of HanesR nylon stockings. The nylon material was sewn to
a 2.5 inch by 2.5 inch piece of foam sponge (packing material from VWR
Scientific, Disposable Pasteur Pipets) and a 1 inch by 1 inch square was cut
from the center of the foam.

The midges were transported to and from the sites in a plastic food
container, lined with damp paper towels. To allow feeding, the nylon bag was
placed slightly anterior to the umbilicus or over the abdominal lesions of each
equine (Figures 13 and 14). The site was rubbed by hand to dislodge any blood
feeding flies, then visually checked before the bag was attached to the animal
with DermiclearR (Johnson and Johnson) transparent tape. The metal clip was
removed and the midges were allowed to feed for 20-30 minutes. After this
time period the metal clip was replaced and the bag was slowly removed;
midges still feeding were collected with the aspirator. The replete midges were
returned to the laboratory and maintained in 10 dram vials as described above
for the engorged flies. The dead flies were removed, fixed in carnoy solution,
and stored in glycerin alcohol over a 23 days period. Flies still alive on the 23rd
day were immobilized by chilling in the refrigerator, then dissected in warm
horse serum (98.6°F). .

The engorged midges in both infection studies were removed from the 5%
glycerin alcohol, then hydrated through descending dilutions of ethanol to 35%
ethanol and stained in Mayer's Acid Hematoxylin (as described above). The

midges were dissected under a dissection microscope and the filarial larvae

39

   

 

nglon stocking

Figure 12 Nylon feeding bag.

40

 

Figure 13. Nylon feeding bag attached to the ventral abdomen
of an infected pony at site B.

41

v”.

 

Figure 14. Nylon feeding bag attached near the umbilicus of the
pony.

42

mounted in glycerol under a coverslip.

FESUJS

E . Q I I I I'

During the period July 14 to October 12 (1984), a total of 36 engorged wild
Quljaajdgg spp. were collected at site A from the ventral abdomen of Lacey
during 4 collection evenings. Three species of Mg: were collected: Q.
gbsglgtug, Q. gtglljfgr, and Q. vguustus (Hoffman). It was found that 2 of 22 Q.
gbgglgtug and 2 of 7 Q. gtglfljgr had ingested microfilariae during feeding
(Figures 15 and 16), but none of the 7 engorged Q. yguggnm were found to be
infected. In addition, a total of 9 engorged wild Q. y. ygnjagnnm were collected
from Lacey on September 7 and 18 (1985) and 4 had ingested microfilariae
(Figure 17).

The results of the skin biopsy confirmed the presence of microfilariae in
the skin of both ponies at site B. Microfilariae were extracted from the pieces
of skin soaked in THS solution (Figure 18). A random sample of 14 microfilariae
were measured and they had an mean length of 224.38 +/- 7.15 u and width of
2.73 +/- 0.32 u. Several morphological structures were frequently seen in the
microfilariae: the cephalic space, nerve ring, rectal cell, and terminal nucleus.
The distance of these structures from the anterior end of the microfilariae
were measured and expressed as the mean distance and percentage of the total
body length (Table 2). Other structures such as the excretory pore, excretory

cell, and anal pore were observed in only a few microfilariaeThey were 66.22

43

II

 

Figure 15. Microfilaria ingested by Q. aggalgtug after feeding
on an ngumgnza infected horse (x 500).

 

Figure 16. Microfilaria ingested by Q. gtgflitgr after feeding on
an infected horse (x 500).

45

l"

 

 

Figure 17. Microfilaria ingested by Q. 1. ygnjaguum after feeding
on the infected horse (x 400).

 

I

Figure 18. Microfilaria from the skin of an infected pony (x 500).

46

Table 2. Morphological dimensions of microfilariae from skin biopsy.’

 

 

Structures Distance from ant. end % distance from ant. end
Cephalic space 3.65+/-0.78** 1.62+/-0.34

Ant. edge of

nerve ring 53.09+/-3.14 23.65+/-0.83

Rectal cell 154.77+/-5.54 68.98+l-1 .14
Terminal

nucleus 21 6.81 +/-6.96 96.63+/-0.70

 

‘Samples were collected from two infected ponies at site B.
“All measurements are in um and expressed as Mean +/- SD.

47

+/- 1.31 u (30.68 +/- 0.45%), n=2; 72.69 +/- 3.06 u (33.52 +/- 1.37%), n=4; and
178.64 +/- 6.66 e (81.69 +/- 1.44%), n=3 respectively, from the anterior end of
the microfilariae. These measurements for the microfilariae are similar to the
results obtained by Collins (1973) and Mellor (1974a) for ngugagma in equines.

Microfilariae were also found in the two pieces of skin submitted for
histopathological examintion (Figure 19). The microfilariae were identified as
an thm sp., probably Q. cgungalm.

W

A total of 1,868 wild Quliggjggs comprising 8 species were collected with
a mouth aspirator from the ventral abdomen of the horse at site A and the
ponies at site B during the 1985 collection period (Tables 3 and 4). However,
four species were commonly found on the horse at site A: Q. gtmglgnm, Q. 1.
militants Q. biounatus (Coquillett). and Q. mutter. Qulimidesobsoletus
was the most abundant species on the horse at site A in May and at both sites in
August, September, and October. At site A Q. mm was the most common
species occurring on the horse in June and Q. gtglljjg: in July. Qujjggiggg
yanlagunm W was most evident on the horse at site A in early May and
again from August to September. The other four species Q. gauggigugg, Q.
ygutmnm, Q. gainggug (Root and Hoffman), and Q. guniaguum (Coquillett) were
collected in small numbers during the collection period.

Filarial larvae were found in Q. gtmglgmg, Q. 1. yafljaguum, Q. stgfljfgr,
and Q. W at site A (Table 5). However, larvae similar to ngugggrga

were found in only the first three species (Figures 20-23). All larvae were in

the thoracic muscles of the Quflggjdgg; none were present in the head or

48

 

Figure 19. Microfilaria in the skin of an infected pony (x 500).

49

 

 

 

 

 

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52

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53

 

Figure 20. Quaugcgrgatype larvae in the thoracic muscles
of wild caught Q. ajmglgnm (x 313).

 

Figure 21. Qucugggrga type larva (broken specimen) dissected
from the thoracic muscles of wild caught Q. gtgmfg; (x 200).

54

I. l
l‘ ,

Figure 22. ngugcgrga type larva dissected from the thoracic
muscles of wild caught Q. g. ygriiagnnig (x 125).

 

L ,

Figure 23. ngugcgrga type larva in the thoracic muscles
of the above Q. 1. ygriiagnnig midge (x 125).

55
abdomen. Several non-filarial type nematodes were found in the thoracic
muscles of Q. gtmglgtug, Q. gtgfljjgu and Q. manatgg (Figures 24-26). No
filarial infections were observed in the Quljgnggg collected from site B.
l . I | I C II I.

A total of 2,134 Quliggidgg and 3 other genera, Egrgiagmyia, Qagyuglga,
and ngzja were collected in the blacklight traps at three collection sites: the
two horse stables in Clinton county (Tables 6 and 7) and the veterinary research
farm in lngham county (Table 8). Five Qulimiggg spp. were commonly collected:
Q. gtmglgtug, Q. 1. yan'iaguu'm, Q. aigunatus, Q. stglljjgx and Q. crgausgulan's
(Malloch). Quligaidgs absalgtus was frequently collected at the two horse
stables thoughout the collection period. At site A this species was most
abundant in early June and from late July to early August. They were found in
small numbers at the veterinary research farm. Quligaidgg yariiagunig
yaniagunjg was collected only at site A and the veterinary research farm; it
was present in small numbers during each month of collection, except for May at
site A. Quliagidgg aigunatus and Q. gtglljfg: were found at all three blacklight
trap sites. Quligaiagg ngausgulang was most abundant at the veterinary
research farm.

EIIIIIIIII I. 'I [GIII

The emergence trap method for collecting Q gagglgtng was discontinued
after a month of operation at site A because only 2 atmglgttm group spp. were
collected during this time period. The two adult midges were collected along the

base of the manure pile. Large numbers of Egmagmyiagjanugtata (Linnaeus)

(Diptera: Ceratopogonidae) were found breeding in the manure pile.

56

 

Figure 24. Non-nguaggma type larva (arrow) in the thoracic muscles
of wild caught Q. gtmglgnm (x 125).

 

 

Figure 25. Non-nguagnga type larva (arrow) in the thoracic muscles
of wild caught Q. stellifgr (x 313).

57

 

 

Figure 26. Non-Qnguoggrga type larva (arrow) in the thoracic muscles
of wild caught Q. blggfianm (x 160).

58

 

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61

In method 2, a six inch garden spade was used to collect samples of
suspected Q. gtmglgnm breeding materials within a 1 mile radius of site A and
B, the DeWitt farm, and the veterinary research farm. In the laboratory, the
samples were placed in white enamel pans, flooded with distilled water. The
pupae that rose to the surface were collected and held in 2 dram glass vials on
wet cotten until the adults emerged. This was an attempt to locate this species
breeding site and to use as a source of clean flies or adults reared from the
pupal stage in experimental ngugggrga infection studies. No Q. gaggjgnm
breeding sites were found, but several other Quflagjggg spp. habitats were
located. At site A, Q. 1. yanjaguum, Q. ngaugguIang, Q. wmgguggugig (Jones),
and Q. afljjg_ri.m group pupae were collected from the wet soil in the south
pasture (Figure 10); Quljgajggggumaguum was found breeding in several
beechwood tree holes in the woodlot; Q. uagmamagtug (Malloch) and Q.
ngagggulang pupae were collected along the margin of a duck pond, 1/4 mile
south of the site. Quliggiags nagmgtgagtag, Q. gmaugginang, Q. ailijgnm group,
and Q. ygutmnm were found breeding along the margins of Summers Drain, 4/5
mile north of site A. A single Qajjagiagg ganjagnum pupa was collected from a
horse water trough at the DeWitt farm about 11 miles from site B. Quljagjugg
mutaguum yaniaguum and Q. grgauggulang pupae were collected from wet soil
at the base of a compost heap adjacent to the Veterinary Research Farm site
(Figure 11).

Experimentauniections
Study 1 was to determine if Q. ggungalm develops to the infective (L3)

larvae in Q. ggsajgjug and Q. y. yanjaguujg after feeding on an infected horse in

62
the field. Engorged wild Q. glmglgmg and Q. 1.1mm were collected by
aspirator from the ventral abdomen of the horse at site A and maintained at
62.6-66.2°F for 25 days post feeding.

Upon dissection, both species were found to be infected with filarial
larvae. Second stage larvae were seen in 2 of 115 (1.7 %) Q. gbgglgtug, 2 and 11
days, respectively, after feeding (Figures 27 and 28). Two of 5 larvae found on
day 2 measured 77.2 and 61.5 u long by 15.7 u wide. The single larva observed
on day 11 measured 143 u long and 15.7 u wide. Both second and third stage

larvae were present in 9 of 54 (16.7%) Q.1.1aflja_gu_u_'m (Figures 29-40).
lnfective larvae (L3) were observed in the head of Q. 1. WIS 6 and 25
days, respectively, after feeding (Table 9). The individual measurements of the
four L3 larvae in the head were: 627, 627, 627, and 684 u long by 18.5, 18.0,

19.1, and 17.9 u wide, respectively. These measurements are within the range
for ngugcgrga infective stage larvae (Steward, 1933; Moignoux, 1952).

In study 2, three replicates of 25 laboratory reared Q. 1. yafijaguum were
fed on Lacey and two replicates on a 15 year old pony named Ginger at site B
(Table 10). Ginger was one of the three ponies examined by Dr. Rosser which had
clinical signs of onchocerciasis.

The first feeding was to determine if the midges would ingest
microfilariae from the area of skin near the umbilicus. It was found that 3 of 7
engorged midges did ingest microfilariae from Lacey and 2 of 2 from Ginger. In
the second replicate,10 Q. 1. yanjagunm fed on Lacey and one on Ginger. Of the

midges that fed on Lacey, developmental stages were observed in 4 specimens

63

 

Figure 27. ngngggrga type larva (arrow) in the thoracic muscles of Q.
gtmglgtug 2 days after feeding on an infected horse (x 160).

 

 

 

Figure 28. Qngugggma type larva (arrow) dissected from the thoracic
muscles of Q. agsglgtug 11 days after feeding (x 125).

64

 

 

Figure 29. ngugcgrga type infective (L3) larva in the head of Q. 1.
variiagnnig 6 days after feeding on the infected horse (x 125).

 

 

Figure 30. Same infective (L3) larva in the proboscis of Q. g. yariiagnnlg (x 313).

 

 

Figure 31. The infective (L3) larva emerging from the proboscis (x 400).

 

 

I
Figure 32. A second third stage infective (L3) larva (arrow) found
in the head of the same Q. y. yau'iaeuuig (x 80).

66

 

 

Figure 33. The infective stage (L3) larva dissected from the head of Q 1.
W (x 125l-

67

 

 

Figure 34. Second stage filarial larvae in the thoracic muscles
of Q. g. variiagnuig 7 days after feeding (x 160).

 

 

Figure 35. Second stage filarial larvae in the thoracic muscles
of Q. 1. yaljjaguum 8 days after feeding (x 313).

68

 

Figure 36. Second stage filarial larva (arrow) in the thoracic muscles
of Q. y. yanjaguum 10 days after feeding (x 125).

 

Figure 37. Second stage filarial larva (arrow) in the thoracic muscles
of Q. 1. yarijagnnis 11 days after feeding (x 125).

69

 

 

I
Figure 38. Second stage filarial larva (arrow) in the thoracic muscles
of Q. 1. lamagnnm 18 days after feeding (x 125).

 

 

 

Figure 39. Second stage filarial larva (arrow) emerged from the thoracic
of Q. 1. yanjaguuig 20 days after feeding (x 44).

70

 

Figure 40. lnfective (L3) filarial larva emerged from the head
of Q. 1. yafijaguum 25 days after feeding (x 80).

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71

 

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72

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73
dissected 4, 6, 14, and 23 days post feeding (Figures 41-46). The single
infective stage larva was found in the head of the specimen dissected on the
23rd day (Figures 44-46). This larva was 627 u long by 19.1 u wide. The single
midge that fed on Ginger died 7 days after feeding and 15 second stage larvae
were present in the thoracic muscles (Figure 47). In replicate 3, only 2 midges
fed on Lacey and 6 second stage larvae were found in one midge 12 days after
feeding (Figure 48). Table 11 shows the development of the nguaanga
microfilariae ingested from the equines at site A and B by Q. 1. yanjagnnjg in

replicates 2 and 3.

74

 

I

 

 

Figure 41. Second stage filarial larva (arrow) in the thoracic muscles
of clean Q. 1. Magnum 4 days after feeding (x 125).

 

J.

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Figure 42. Second stage filarial larva (arrow) in the thoracic muscles
of Q. 1. yanmgnnm 6 days after feeding (x 125).

75

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Figure 43. Second stage filarial larva (arrow) in the thoracic muscles
of Q. 1. yaniagnum 14 days after feeding (x 160).

 

 

‘I

Figure 44. An infective (L3) larva in the head of Q. 1. ganjagunm
23 days after feeding (x 50).

76

 

 

 

 

 

Figure 45. lnfective (L3) larva emerging from the proboscis
on.v.1ampanni§ (x 200)-

77

 

 

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Figure 47. Second stage larvae in the thoracic muscles of Q. y.
yanmgnnm 7 days after feeding (x 160).

78

L_

 

Figure 48. Second stage larvae (arrow) in the thoracic muscles of Q. g.
magnum 12 days after feeding (x 160).

79

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DISCUSSKN

The original purpose of this study was to evalute the role of Q. W
as a potential vector of W in equines, but Q. 1. yaflipmmis was—later
included in the study for two reasons: 1) It was observed during the second year
of the study that wild caught Q. y. W midges were repeatedly found
infected with microfilariae after feeding on the horse at site A; 2) two breeding
sites of Q. 3;. yadjnamis were found and moderate numbers of larvae and pupae
were collected from these sites, so it was an ideal opportunity to determine if
anhocmca species would develop to the infective stage in this subspecies of Q.
W after feeding on an infected equine in the field.

According to Barnett (1960). four criteria must be demonstrated to
incriminate an arthropod as a vector:

1. Demonstrate that the suspected arthropod feeds on or makes
effective contact with the host under natural conditions.

2. Demonstrate a biological association in time and/or space between
the suspected arthropod vector and the occurrence of clinical or
subclinical infection in the host.

3. Repeatedly demonstrate that the suspected arthropod vector
harbors the identified infectious agent in the infective stage under
natural conditions.

4. Demonstrate the transmission of the infective stage to a susceptible

80

81
host under controlled conditions.

Previous authors have reported Q. Mus feeding on equines in the field.
Steward (1933) collected engorged Q. 91259191115 from horses in England and
Shemanchuk (1972) reported that this species bites horses in Canada.
Schmidtmann et al. (1980) collected them from ponies in New York. According to
Blanton and Wirth (1979), Malloch in 1915 reported Q W biting horses
in Illinois. This northeastern subspecies of Q. yafljpgnnjs is considered to be Q.
1. W. in this study, both Q. 1. mm and Q. mm were
collected by mouth aspirator from the infected equines at site A and B (Tables 3
and 4). This demonstrates Bamett's first criterion for incrimination of a vector.

The second criterion requires proof of biological association between the
suspected vector and occurrence of clinical infection in the host. According to
McMullen (1972), clinical signs of equine onchocerciasis are common from
spring to late autumn. These signs include loss of hair and the presence of scaly
skin lesions on the body of the equines, particularly on the head, face, chest,
neck, and ventral abdominal midline; there may be mild to severe itching
associated with the infection. These conditions usually improve during the
winter, but reappear in the spring. The duration of mm infections in
equines is unknown, however, Mellor (1973a) recorded Q. mu:
microfilariae in the skin of horses from June to February in England. McMullen
(1972) reported that adult Q. 191m lives for 10-15 years in man. Clinical
signs of onchocerciasis in equines are not evident in all infected equines so a
skin biopsy or other means of demonstrating the presence of microfilariae is

necessary for confirmation of infection.

82

The horse at site A was used for breeding and appeared to be in relatively
good health. Clinical signs of onchocerciasis in that horse were first observed
on July 14, 1984 and during the month of June in 1985. These signs included
thinning of the hair along the midline of the ventral abdomen, extending from
the xiphoid process to the umbilicus and a single skin lesion about 1 inch in
diameter lying midway between the forelegs. This horse was not biopsied, but
engorged wild Q. Mus and Q. 1. yafiipgnnis were collected from the ventral
abdomen of the horse and later dissections demonstrated several apparently
different types of microfilariae in their blood smears. The blood smear prepared
from Q. W was stained with Giemsa and three microfilariae were
observed in the blood smear. Two of the microfilariae were about the same
size,195 u long by 2.5 u wide. They were within the size range of Q. mus
microfilariae stained with Giemsa by Mellor (1974a). The other nematode was
broken during the preparation of the blood smear; it was smaller about 139.8 u
long by 3.1 u wide and lacked a cephalic space at the anterior end. This nematode
was not within the size range for Qnghma. Two types of microfilariae were
also observed in a hematoxylin stained blood smear from an engorged wild Q. 1.
mnenms that had fed on this horse. These microfilariae were fixed and
stained using the method described by Collins (1973). The measurements of the
two microfilariae were about the same size, 214.4 u long by 2.5 u wide and
218.7 u long by 3.1 u. The cuticular striations in the latter microfilaria were
more evident under oil immersion than the former microfilaria. It is assumed
that Qnmgema species are host specific and the species occurring in equines

in the United States is Q. Wis. Both microfilariae in the blood smear from

83
Q. 1. yafiinemjs were considered to be Qngmgema, since their measurements
were within the size range of Q. mus reported by Collins (1973). At site B
abdominal lesions were observed in 3 of 5 ponies. Biopsy samples taken from
the lesion and umbilicus area of the two ponies contained Qnghmma
microfilariae.

The results of the blacklight trap and horse baited collections showed that
both Q. obsoletus and Q. 1W were found on or near the equines at
sites A and B throughout the 1985 study period, except that Q. 1. W was
not collected during the month of August at site B. In addition, Q. 1. yaflipennis
was found breeding in the south pasture and limited breeding of a Q. Mus
group species was found in the west pasture at site A. Several potential
breeding sites for Q. cm: were located near the site a pond, the Watson and
Summers drain, and a large marshy area located 400 ft from the western edge of
the site. Wet leaflitter and soil samples were collected from the several sites
along the margin of the marsh and the Watson and Summers drain, but no Q.
mm were found. Time limitations in the study did not allow a more
detailed survey of these areas for Q. W. No breeding sites for Q.
chaplains or Q. 1. W were found at site B, but potential breeding
habitats such as a corn field and marshy area, drainage ditch, and manure pile
were located nearby. The results of the biting and light trap collections and the
origin of these species in the vicinity of the collection sites satisfy Barnett's
second criterion for vector incrimination.

The third criterion states that the suspected vector must repeatedly be

found to harbor the identified infective stage under natural conditions. Unlike Q.

84
191mm, there have been few detailed descriptions of equine Qnghggerga larvae
in the arthropod host. The identity of equine Qaghm larvae has been based
on their size, site of development of the larvae and the assumption that the
species occurring in the skin of equines are Q. cmigafls and Q. m.

Steward (1933) followed the development of Q. migafls to the infective (L3)

stage in engorged field collected Q. W in England; they measured
600-700 u long by 18-21 u wide. Foil et al. (1984) reported finding a third stage

Q. M larva in a wild Q. W collected in a pony baited stable trap

in Louisiana, but they did not indicate it's measurements. Bain and Petit (1978)

redescribed the infective (L3) larvae from laboratory infected Q. W in

more detail and reported that the infective (L3) larvae ranged in length from

680 to 870 u, the longest measured 870 u long by 18 u wide. They also listed
measurements for several internal structures: buccal capsule, nerve ring,
excretory pore, oesophagus, intestine, rectum, and tail. Wild caught Q. chaplains
and Q. 1. mm at site A were found infected with filarial larvae in the

thoracic muscles. Several different types and stages of larvae were observed in

the thoracic muscles of Q. Mus, but no Qnghm infective (L3) larvae
were observed. The size and shape of most of the larvae in Q. m did not
resemble the developmental stages of Q. was described by Steward (1933)
and Mellor (1975). However, three larvae were found in the thoracic muscles of
Q. Mus which did resemble first stage Qnghggma larvae in appearance and
body width, but their average size, 112.97 u long by 5.72 u wide (n=3) was below
the size range for Qngmggma. Two second stage anhncenca type larvae were

85

found in 1 of 37 (2.7%) wild caught Q. 1. variipennis. A late second stage
Qngngggma type larva was found in 1 of 322 (0.3%) Q. mum. This species
was the predominant species occurring on the infected horse in July during 1984
and 1985. In addition, non- W larvae were found in the thoracic
muscles of 2 Q. My: specimens dissected. It is possible that these
non-QnQangma type larvae found in the thoracic muscles of Q. Mus, Q.
519111191. and Q. m may be from a non-equine host. Lichtenfels (1975)
listed 6 non-equine filarial larvae found in the horse. According to Linley
(1985), ceratopogonids are vectors of 17 filarial nematodes of which 8 are bird
filariae. They may also be the larvae of a parasite of Quljggidgs. Mermithid
parasites have been reported from adult Q. 91259191115 (Service, 1974). According
to Buckley (1938), mermithid infections in the thoracic muscles of Quliggjges
resemble filarial "sausage" stages.

In experimental infection studies, filarial nematodes were present in
engorged wild caught Q. 9mm and Q. 1. yafijpgmjs after they had fed on the
infected horse at site A. Second stage larvae were found in 2 of 115 (1.7%) Q.
M91115- These larvae were in the thoracic muscles 2 and 11 days post feeding.
Although the larval stages were similar in shape, their size was below the
range for W. Because of a lack of experimentally infected flies for
comparison, it was not possible to determine if these larvae were ingested from

the skin of the horse or had been acquired from a previous blood meal. lnfective
(L3) larvae were present in the heads of 2 engorged wild caught Q. y. lan'inennis

6 and 25 days after feeding on the horse at site A. The lengths of the infective

86

(L3) larvae measured 627, 627, 627, and 684 u, their widths were 18.5, 18.0,

19.1, and 17.9 u, respectively. The length of these infective (L3) larvae were

within the size range of W (Steward, 1933). The presence of third

stage larvae in the head of QuleQldQQ indicate that they are infective and able

to transmit the infective (L3) larvae to the equine host during feeding.

The infective (L3) stage of Qnghgggma also developed in laboratory reared

Q.v.1amp_em1i§ allowed to feed on an infected horse at site A (Tables 10 and
11). The horse was exposed to a total of 75 Q. 1. yafimannis and 19 fed. A single

infective (L3) larva was found in the proboscis (lying between the mandibles) of

this midge 23 days post feeding and the larva measured 627 u long by 19.1 u
wide. Since this larva was similar in appearance and within the size range of Q

m reported by Steward (1933) in English horses , it was assumed to be

that species of Qnghggerca. The finding of infective (L3) larvae of Qngmcema

sp. in wild caught and wild and experimentally infected Q. 1. mums at site
A satisfy Barnett's third criterion. Three out of 50 of these flies fed on the

pony at site 8; they were dissected 0 and 7 days post feeding, but no infective
(L3) larvae were found.

The fourth criterion, demonstration of transmission of the infective stage
to a susceptible host under controlled conditions was not attempted in this
study. The lack of a susceptible host other than equines and the cost of utilizing
these animals in controlled studies prevented the complete incrimination of

Qujjggjdgs as a vector of Qngnmfi in this study. Collins and Jones (1978)

87
allowed infective Q. yadjnannia to feed on horse semm using a membrane feeder
and observed transmission of Q. Mafia infective larvae into the horse serum

solution. They also fed the infective Q. W on jirds (Marianas,
unguicujatua), and dissected samples of midges before and after feeding on the

jirds to determine the number of infective (L3) larvae in the flies and how many

were lost during the feeding process. In addition, the skin surface of the jirds
were examined and no larvae were seen. From the results of their study they
concluded that 62.5% of 369 midges shed all their infective larvae while feeding
on the jird. No mention was made of whether the infective larvae continued
their development in the jirds.

Based on the findings in this and other studies on the transmission of
Qnghgaarga by Qujjaaidaa, it is concluded that Q. 1. milpannia is a potential
vector of equine onchocerciasis in Michigan and that Quliggjdaa atafljier may be
a potential secondary vector. The role of Q. W as a potential vector is
still unresolved, since no Qnahgaama infective stage larvae were found in
several hundred dissections of wild caught midges. This is not uncommon since
the natural infection rate of Qnamgaraa in Quliagjdaa species is quite low. In
Malaysia, Buckley (1938) found that only 13 of 3,734 (0.35%) Q. magma (de
Meij) were naturally infected with Q. gjnagni (Cleland and Johnston), a bovine
parasite. In France, Moignoux (1952) reported that 2.1% of wild caught Q.
M were naturally infected with Q. m. In this study, natural
infection rates of 2.7% and 0.3% for Q.1.1a[j_|,p_9_n_nj_a and Q.a1_e_[1j_ta;,

respectively were found.

WWW

mm infected equines were diagnosed by demonstrating the
presence of microfilaria in blood taken from engorged Quligmdaa and
in skin biopsies from suspected equines.

Wild caught Q. am and Q. 1. mm were collected from
infected equines at both study sites.

Natural infections of Qngmzaama were found in 1 of 37 (2.7%) wild
caught Q. 1. mmma and in 1 of 322 (0.3%) Q. stalling; however, no
infections were observed in wild caught Q. gnammua.

Qnahgaama infective stage larvae were found in the head of both wild
and experimentally infected Q. 1. yadjpamia midges.

Using the four criteria described by Barnett (1960), it appears that Q.
1. mmmnma is a potential vector of equine onchocerciasis in
Michigan and that Qujjggidaaatalflm may be a secondary potential
vector. The role of Q. W as a potential vector is still
unresolved, since no szhggmaa infective stage larvae were found in

several hundred dissections of wild caught flies of this species.

88

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