Quote from full text on the subject of the microscopic examination of ticks for the Lyme disease spirochete:
"In late September and early October 1981, Dr. Benach provided
additional collections of I. dammini from Shelter Island, New York,
where Lyme disease was known to be endemic. Again, none of 44 males and
females had rickettsial infections. The hemolymph of two females,
however, contained large microfilariae that differed morphologically
from Dipetalonema (Wehrdickmansia) rugosicauda, a microfilaria detected
in several adult 1. ricinus in Switzerland in 1978 (18).
To determine whether these nematodes were present also in the digestive
system, I dissected both ticks and prepared Giemsa-stained smears from
individual midgut diverticula for microscopic examination. No
microfilaria was found. Instead, I encountered poorly
stained, rather long, irregularly coiled spirochetes (Fig. 1).
Darkfield microscopy of additional diverticula confirmed the
spirochetal nature of the organisms, which had rather sluggish and slow
movement. Additional tissues, including salivary glands, malpighian tubules, ovary, and central ganglion of either tick, were free of spirochetes.
Subsequently, 124 remaining ticks were dissected and each of their organs was examined for similar organisms. Seventy-five (60 percent) contained spirochetes that were limited to the midgut (Fig. 2).
Organisms occasionally seen in preparations of hindgut and rectal ampule may have originated from midgut tissues. All other tissues were free of spirochetes.
Remembering the European literature, I could not dismiss the thought
that the microfilariae did lead me to the discovery of the long-sought
cause of ECM and Lyme disease.
Needless to say, I shared these observations with several of my colleagues, including Dr. Benach
who not only saw to it that I was well supplied with field-collected
ticks from Shelter Island, but also provided sera from patients with
clinically diagnosed Lyme disease for preliminary serological
identification of the organism, and also Dr. Barbour
who at the time was engaged in a study of the variable major proteins
of cultured tick-borne relapsing fever spirochetes and who offered his
expertise to culture and immunochemically characterize the organism
. [for more on the latter subject see http://lymerick.net/Borrelia-culture.html
The antigenic relatedness of the I. dammini spirochete to the etiologic agent of Lyme disease was established by indirect immunofluorescence
as well as by western blot analysis of sera
from both Lyme disease and ECM patients
[19,20]. Our initial indirect evidence that this organism might be the cause of this disease was subsequently confirmed by isolating from patients spirochetes indistinguishable from those detected in I. dammini and by the microscopic demonstration of spirochetes in skin biopsies of cutaneous lesions of several Lyme disease patients
One of our subsequent research objectives was to determine whether I. ricinus
the incriminated vector of ECM in Europe, was also a carrier of
spirochetes. Evidence that this was the case was obtained from smears prepared in 1978 from nymphal ticks at the University of Neuchâtel in Switzerland
, where I spent several months conducting a tick/rickettsial survey in various parts of that country. Of
135 smears of ticks collected in the Seewald forest on the Swiss
Plateau, where according to medical authorities ECM had occurred in the
past, 23 (17 percent) contained spirochetes
that tinctorially and morphologically appeared similar to those detected in I. dammini.
In the spring of 1982, the U.S. Department of Agriculture gave us
permission to import about 600 adult I. ricinus from the Seewald forest
(201 of 201 individually examined ticks, 73 (36.3 percent) were infected with spirochetes. The organisms were limited to the midgut in 69 ticks but were found in all the tissues of the other four ticks. [4/73 = 5% were SYSTEMICALLY INFECTED!]
Of an additional 180 females that were fed on rabbits, 39 (21.9
percent) were infected. Two of them [2/39 = 5%] had a generalized
infection whereas the other 37 had spirochetes in their midgut only.
Both females with
generalized infections transmined spirochetes via eggs to 100 percent
larval ticks in one and 60 percent in the other.
However, as the
larvae developed to nymphs and adults, the degree of spirochetal
infection gradually decreased to a level of few organisms in tissues of
the central ganglion only. This spirochetal behavior is in sharp
contrast to the massive and prolonged development of tick-borne
spirochetes and suggests that the growth conditions in the hemocoele of
I. ricinus are inferior to those in the midgut.
Morphologically, the I. ricinus
spirochete appeared indistinguishable from the I. dammini organisms,
and antigenic similarities between the two spirochetes were apparent by
direct immunofluorescence and SDS-PAGE protein profiles as well as by
indirect immunofluorescence and western blot analysis of sera from ECM
and Lyme disease patients (20).
Convinced that ECM of Europe and Lyme disease in the United States are
expressions of one and the same etiologic agent, we directed our
attention toward the west coast of the United States
where the first case of ECM was contracted in Sonoma County, California, in 1975
and where the black-legged deer tick, I. pacificus
, had been incriminated as the vector.
In collaboration with Dr. Robert Lane from the University of California
at Berkeley and Dr. Robert Gresbrink from the Oregon State Health
Department in Portland, we initiated a tick/spirochete survey in south-western Oregon and in north central California
— areas where I. pacificus is abundant
The test procedures were similar to those applied to I. dammini and I.
ricinus. Adult ticks collected by flagging vegetation in the spring and
early summer were dissected individually, and the midgut diverticula
were smeared on a microscope slide. After air-drying and after
ten-minute fixation in acetone, the smears were treated
with FITC conjugates prepared from sera of New Zealand white rabbits
that had been immunized with the Lyme disease (Shelter Island isolate)
[i.e. DIRECT FLUORESCENT ANTIBODY STAIN, as later
used by others to stain spirochetes in culture and in blood from Lyme
Ticks with spirochetes in their midgut were further dissected to determine the presence of organisms in other tissues.
Of 645 I. pacificus from Oregon and of 550 ticks from California, 13 (2 percent) and 5 (0.9 percent), respectively, contained
spirochetes that morphologically and by fluorescence microscopy
appeared indistinguishable from the spirochetes found in I. dammini and
I. ricinus. Five of
the 13 [5/13 = 38%] infected ticks from Oregon and two of the five [2/5
= 40%] infected ticks from California had a generalized infection
the remaining positive ticks had spirochetes only in the midgut. As
yet, we have not succeeded in establishing in modified Kelly’s medium
an isolate for immunochemical analysis. The low percentage of
spirochete-infected ticks in the West certainly is reflected by the
small number of Lyme disease cases reported so far. It is quite
possible, however, that there exist, within the distributional areas of
I. pacficus, foci with higher infection rates providing a greater
potential for human disease.
In concluding my presentation, I would like to emphasize that studies
pertaining to the relationship of Lyme disease and ECM spirochetes to
their respective tick vectors are still in the initial phases. As yet,
little is known about the development of these organisms in ticks and
about the mechanics by which they are transmitted to hos animals and
man. Nevertheless, the observations and findings discussed above permit
the following conclusions:
1. Spirochetes antigenically similar, if not identical, to each other
have been found associated with Ixodes ricinus, I. dammini, and I.
pacificus, the currently known tick vectors of ECM in Europe and of
Lyme disease in North America.
2. Spirochetal development in most ticks is limited to the midgut. In a
few ticks, the organisms penetrate the gut epithelium and invade the
hemocoele and various tick tissues. Their presence in the ovary may lead to transovarian transmission
Nevertheless, there is evidence that spirochetal infection in filial
ticks decreases and becomes restricted to the central ganglion as the
ticks develop to nymphs and adults.
3. It is speculated that transmission of spirochetes during the feeding process may occur via saliva by ticks with generalized infection as well as by regurgitation of infected gut content.
Contamination by spirochete-infected fecal material cannot be excluded.
4. Finally, in view of the unusual behavior (limited distribution, loss
of infection intensity) of the spirochetes in ticks, the possibility
that other hematophagous arthropods, such as biting flies, gnats, and
mosquitoes, may play a role as mechanical vectors of ECM and Lyme
disease cannot be ruled out."
This study shows that B.
burgdorferi can be
maintained through transovarial and subsequent transstadial
transmissions in I.
Borrelia varied, in
nymphs (n = 347) from 13% to 46% and in adults, (n = 122) from 20% to
in larvae (n = 84) collected in 1993 was 21%,
showing that transovarial transmission of B. burgdorferi occurs in the
I. ricinus population on Ameland.