Trypanosomiasis and Animal Agriculture: A Study of...
Trypanosomiasis and Animal Agriculture:
A Study of Pertinant Regions
and Strains in Senegal
. -
by Crystal Waters
Intemship 1998
TSRA: Dakar, Senegal
Advisor: Dr. A. Diaite
Table of Contents
Introiluction
3
Literar y Review
5
The Trypanosome
The T\\:ector
Main 1,aborator-y Projet;
11
Introduction
Ol+ctives and Approach
Methods and Techniques
Results and Interpretations
Discussion
Seconuary Projects
1 7
Inducing trypano-tolerance
Wild rats
Separation of trypanosomes from hlood
Cloning
FielcI \\X’c,rk
23
Dairy Farrns in the Niayes
Par; Bandia
SotGMe
2 8
37
Introduction
“Trypanosomiasis of man is surpassed only by malaria as an infection that inhibits the
development of tropicallands”. l” It causes the suffering of ovl:r one million Africans (20,000 :I~:\\V
cases each year) and severe malnutrition due to the sickness and loss of livestock. i Trypanosomiasis
was first described in the fourteenth Century in what is IOW thc. country of* Mali. By 1902 two
English physicians in the Gambia ascribed the causative agent to be a parasite, and one year late;
another scientist identified the tsetse fly as the vector.” In 1906 Winston (‘hurchill stated that thc
population of Uganda had been reduced by sleeping sic,kness fi-om 6.5 to 2 5 million.4 The samc year
Ayres Kopke introduced Atoxyl, an arsenic compound for tre:ltment of thc disease, and by tht: carly
1950’s human sleeping sickness had been greatly minimized through surveillance programs and
vector control.‘4 However since the 1960’s trypanosomiasis has been on thf rise in Africa, as p:)st
independance social umest and lack of fùnds has causeCI many control and tleatment programs t. be
neglected..“8 Today sleeping sickness occurs in 36 Afrlcan coi.ntries in which more than 50 million
people are at risk.
Animal sickness and mortality due to trypanosomal infections ha\\,e placed even greater
constraints on development in tropical lands. Anemic animals nnpedt: agriculture development atld
contribute to severe malnutrition There are fifty times as many pathogenic jpecies that affect
animals than do humans, and some of these are transmitted in itreas uninfected by traditional vectors,
for example the tsetse fly. In Sub-Saharan Africa the annual loss due to animal mortality and
sickness was reported in 1991 to be approximately (US) $4 billion, representing 24% of the total
livestock production.‘3
This is particularly detrimental as the continent’s population is increasing hy
3.1% each year and severe malnutrition calls for an inc; ease, net decrease, of animal production ln
an effort to improve public health, the World Bank set .i goal for the continent of Africa of an aIrrua
food production growth rate of 4% by the year 2025.i7
This will only be possible with a containment
3
of the critical animal diseases. Trypanosomiasis is a particularly important disease as animals cari
produce partial resistance for several months to years, tiuring which time the animal is alive but
anemic and often unable to procreate or have good productivity levels.
Senegal is a country with variety in climate, ranging from the arid north to the sub-hum4
-,: *
south and south-east. Much of the state would be considered semi-arid, in which low rain fa11 is
particularly suitable for animal raising. The western Niayes valley just north of Dakar is well knolvn
for it’s potential as a very profitable farming region. Tire area \\\\;as infested with tsetse flies until t11e
early 1970’s when a control campaign was implementeti This was followeti up in the 1980’s with a
second phase which included insecticide impregnated traps as well as the prcvious method of
ground-spraying.’ However since mid 1997 tsetse flies have been reported in various parts of thc
region and every rainy season farmers in the Niayes report cases of trypanosmiasis in their herds
(most often 7:vi~ux). Yet whether tsetse flies alone or other biting flies as well are responsible is not
clear as no fly serveys are being carried out. Most of tht: technologically advanced farms havf:
implemented seasonal animal-spraying programs to reduce the incidence oi‘vector-born disease:;
Considering the importance of the Niayes regicn in Senegal, nejv surveillance and contrc:l
programs are being implemented by organizations such as FAO. The main project completed w Ilris
intemship was using a strain of trypanosomiasis recently recovered from a symptomatic cow in
Bambilor; located in the Niayes valley. The focus of the study was on the virulence of this strain in a
variety of hosts, ruminants and rodents. Other smaller projects were completed during the courst’ of
the internship and are included in the report as they adlled to the comprehension of the study
A I~W
field visits were also made to trypanosomiasis-infested areas in Senegal for observation oftsetse-host
interaction in the natural environment.
4
Literary Review
The Trypanosome
The trypanosome is a protazoan parasite in the class mastigophora As of 1972 there w:xe
over one hundred subgenus identified, classified in two main groups: Stercfx-aria and Salivaria
(infection via feces or saliva, respectively). The subgenus in Stercoraria are far more numerou;,
although those in Salivaria are often more pathogenic (Figure 1). The trypanosorne’s physical
morphology varies greatly among subgenus, although the basic structure is univcrsal (Figure 2)
kinetoplast.
A”
‘\\
undulating membrane
flagellum
(Figure 2)
The largest trypanosomes are found in the subgenus kfegatrypamrm and cari be as long as 13Oum
(T.ilzyerzs) and 15um wide (1Itheileri), whereas the smaller Nutmomorma.s
crin be as short as 8c~r11 iu
1ength.7 The body surface of the trypanosome is a fairly strong membrane, known as the pelhcle OI
periplast, which consists of three layers. Underneath the pellicle lies a network of nearly 100
microtubles which help to maintain the shape of the parasite. The parasite has a single flagellunr
originating in the kinetoplast, enabling it to swim freely within the host’s bloodstream. The lengt 11 of
the flagellum varies greatly among the different subgenus of trypanosomes. The use of biornetl: a>
well as the behavioral pattems in fresh blood cari help in identifying the subgenus of a parasite.
Some are very quick and traverse a microscope field with incredible speed (7:vivcrx) whereas otlters
appear very lethargic and even motionless at times (ïIJzris).7 Three different species were studied in
lab during the course of the internship: 1: vivax, 7: coppIet~.~e, 7: lewisi.
5
be uninfective to rodents but has been found in ungulates such as sheep, goats, and camels with
bovines being the principle host7 Pathogenicity varies, from Nigerian Zebu cattle who have an
untreated mortality rate of 100% at?er 3-4 months of exposure to some cattle in the Congo which
have been known to spontaneously recover alter 2 months of a Tvivax infection. Both wild and
.,1 _:
domestic ruminants are susceptible, with periods of incubation varying from 4-2 1 days for virulent
strains and up to 59 days for mild cases. Parasiternia ?:vels fluctuate g-eatly, even within one day,
although parasites are often more numerous in the first few months of infectlon
i?congolense is in the subgenus Nannomonas, often lacks a free flagellum, and is usua!l~,
smaller (8-24um) than Tvivax (Figure 3b) ‘/:cor~~o/t?~~ may be one of thc most econmically
important species of trypanosomes because “a11 species of domestic mammals are susceptible to
infection”.7p445 Within the many subspecies of congolense there is an apparant trend of greater
pathogenicity with the longer forms. Although 1:congolerrse kills most hosts within 6- 12 weeks
afier infection, there are reports of spontaneous recovery. Laboratory animais (including rodents)
are easily infected, although occasional strains require ser-ial passages from the original natural host
Figure 3
Host resistance to both Tvivax and ï:cmplerrse infection is greatly improved by good
nutrition and sanitary conditions. The N’Dama cattle (an indigenous breed ti, Africa) are weli krlown
to be tolerant to both Tvivax and Kcorrgolensc yet immunity may be an acquired trait, as cattle with
extended unexposure show greater susceptibility.7 On the other hand, Zebu cattle (originating in
Asia) show high levels of susceptibility regardless of exposure to trypanosomiasis.
The third species studied in lab is ?Yervisi which is in the fecal transmitted group of
Stercoraria. The average length of Tlewisi is 26-34um and on.3 of the distinguishing factors is a
7
(Subgenus) Megatrypanum
T. ingens (ruminants)
T.p(fami (bats)
. ..32
Hupe tosoma
T. lewisi (rats)
..‘; _.
T.prinmluni
Group Stercoraria
T. rarigeli
. ..53
1 Schizotrypcnmm
7: cr~zi jman)
T. hipposideri
. . 9
Genre Trypanosoma
7: viwx [ruminants)
Group Salivaria
II(7 :) b. brucri
1: (‘I~)b.gamhicil.sc
(mari)
densierlsct (~11s n)
Figure 1
T.vivux is in the group Salivaria, subgenus Duttonella and its size cari range from 18-3 1 )II~I
(including flagellum) with the majority being 20-26pm.’ The distinctively large kinetoplast (diameter
1.1 pm) and long free flowing flagellum help when identifiing 7: vivax (Figure 3a). Multiplication IS
via binary division and is initiated at the kinetoplast. 7: vivax is “xe of the most important and
widespread forms of trypanosomiasis of livestock in tropical Africa”.7 p “’ The strain is believed lo
6
rather long, rigid, and pointy posterior end; whose distance from the kinetoplast is longer than found
in 7:congolense or T.vivax (Figure 4). The principle (and perhaps only) host of Tlewisi is rats, most
commonly in Black and Brown rats which are found all over the globe.7 Incubation is approximately
6 days, after which the parsite undergoes rapid multiplication until the host’s immunity responds,
killing many of the parasites, leaving only adult forms in the blood. In the majority of cases, ~:~WV.YI
is nonpathogenic although pathogenicity has been attained through rapid serial passages7 In 193 3
one case was reported in a Malian Child whose home \\I’as infested with infected rats7
Figure 4
Although the trypanosome is one of the most studied l)rc)tzo;m parasites, it has eluded the
scientific development of an effective, long term vaccine. As Salivaria species are the most
pathogenic, study has been focused on the few species that are found within that group. One of the
more difficult factors is that the parasite evades the immune response by changing its external antigen
coat every few days. Just as a host has begun to identify and fïght the infèction, the parasite changes
outfits and runs rampant again. l” This scenerio produces peaks of parasitemia, much like the
oscillating temperature of a malaria patient. Trypanosomiasis is also dificult to study because the
parasite is rather fragile outside of a live host environmznt. In the late 1980’s scientists were st.ili
working on suitable growth medias which do not require feeder-cells for the infective form of Lhe
parasite.z6 Earlier studies claimed that the crucial ingredient was cysteine whereas the Baltz and
Hirumi lab groups have found animal serum and reducing agents (thioglycerol or 2-mercaptoethanol)
to be the determining growth factors.2’6
8
As well as the detailed studies mentioned above, more fundamental and observational
research is being done on control methods and vector-host relationships. The late diagnosis of‘an
American safari tourist (9 weeks afier exposure) with Ithodesizn trypanosomiasis reveals the
dificulty of identifying the disease, and extent of unawareness in the United States.” In the earlq
i’. . .*’
1990’s in Ethiopia, the Leak group found that insecticide-impregnated targets were effective for the
control of tsetse flies and trypanosmiasis in cattle herds.(’ While observing the behavioral patterns of
vector-host interactions in the Central African Republic (tsetse flies with pigs and humans), it 1~2s
concluded that a low densitiy of flies led to a greater proportion of human blood meals. ’
The Vector
The cyclical vector of Salivarian trypanosomes is the tsetse fly (~lossirrn. Glossines are long,
brownish black flies ranging 6-16 mm in length and easily identified by two distincive characteri:;tics,
crossed wings when sitting at rest and the cellule discale in the extended wing which is hatchet
shaped instead of the normal triangle (figure S(a)).
(a) cellule discale
Al1 30 species and subspecies of Glossina are present only in Sub-Saharan Africa, two of which are
found in Senegal: Glossina palpalis palpalis and Glossina morsitans submorsitans. G.pa1pali.s and
G.morsitnans are distinguishable most easily by their leg coloring. On the C;.pdpdi.s a11 five sections
on the leg are black (stockings), whereas only the last two on G.mar.sitcm.s (socks) (Figure 5(b)lI
9
Tsetse flics do not require blood meals every day, although on an empty stomach a fly cari
take up to its own weight in blood (155 mg) within 25 seconds. At the beginning of a blood mcal
saliva is deposited into the host to reduce blood coagu!ation and it is during this time that a great
quantity of trypanosomes cari be transmitted. One of the most scientifically important factors of thc
‘i .,
tsetse is their unique reproductive systems. Similar to the flesh-eating screwworms, female tsetse
flics are only fertile at the beginning of their lives because the reproductive anatomy is such that afier
an initial mating no more sperm cari enter. During the life of the fly, semen is delivered into the
uterus for fertalization. This one-time mating reproductive system has led scientists to develo;,
control programs using sterile males. Unfortunately, releasing sterile male tsetse flies has its dra\\~
back as they temporarily increase the possibility of disease transmission (unlike screwworms because
the male is harmless).
There is evidence that some species of Salivarian trypanosomixis cari also be transmitte+
mechanically by other biting flies, such as Stomoxyine and ‘làhirlidea. Unlike with Glossincr, u.:~icll
play a developmental role in the life cycle of the parasite, mechanical transfer is more like a
laboratory infection with a syringe. Such transmission must be quick (3-5 min), as success depeqds
on the survival of the parasites on the mouth parts of the fly. 7irbinide.s are particularly menacinS. as
their saliva has an anticuagulate that causes hemoraging at the site of bite, yet unlike Glossir~a and
Stomoxyine, only the females are hematophages (blood eaters).
The last species of trypanosome studied in lab was T.lewisi, which is cyclically transmittcd by
the rat-flea, Xenopylla cheopis. However, “a number of observers have demonstrated that this
trypanosome cari also develop in other fleas’17 p227 such as those that attack dogs, mice, or hur;ans
The development cycle in the tlea is about 5 days, aRer which they are discharged in the feces of the
flea, infecting a new host at the site of the bite
10
Main Laboratoxy Proj ect
Introduction
In early Septe@.er, 1997 a small farm owner in the Bambilor region complained of animal
sickness and death in cattle and goats. Blood from one of these animals was taken to Dakar where
laboratory tests (fresh blood smears) revealed a well progressed 7Ivivax infection. Prior to
analyzation, five mice were injected with the infected blood. A few days later the death of one I )t
the mice led to the examination of the remaining four, one of which showed positive parasitemia
The infection was monitored daily and fluctuating parasiternia was observed in one of these mtct’ f’~,l
over one hundred days (Annex A. 1). A single passage given to five other mice (two naive) produced
parasitemia in four, the longest of which lasted over sixty days.’
T. (‘ttorzella) vivax was first identified by Ziemann ( 1905) in domestic ruminants and ovtx
the past Century a vast number of T.vivax strains have been discovered, none of which “was nclrmally
infective to rodents.” 7 @03 If the species is indeed isolated to ruminants, then epidemics cari be
avoided by quick treatment of symptomatic animals. This has been very helpful in the Niayes regiol:
of Senegal, where T.vivax is known to be a problem. As mentioned previously, there is evide,lce that
Zvivax cari be mechanically transmitted by biting flies other f.han Glossina. The possibility that a
strain is transimissable to un-immunosuppressed rodents poses a threat to containment programs
The parasitemia observed in the fïve mice directly infected from the Bambilor cattle wus
notable in three ways: long duration, high level of para:;itemia, and non-pathogenicity. All of’ thesc
factors contribute to making rodents very good intermediary hosts, both between farms and also as
an uncontainable animal reservoir during non-endemic periods (ie Senegal’s dry season). As fiir:her
investigation of the Bambilor strain was needed, this internship was the follow-up experiment with
the following hypothesis: in the Niayes region, a strain of ‘/: (I.hrtto~rella) vivax is transmissible and
infective to laboratory rodents.
II
Objectives and Approach
The objective of this project was to confirm the results of the previous experiments (which
showed high levels of parasiternia in 83% of the infected rodents) With a larger group of animais
than previously used (Group A: 10 mice, 1 rabbit), the rodents would be infected with the Bambilor
T. vivax strain and daily observed for parasiternia. When the parasite Count was high enough, the
first serial passage would be done, (group A2: 10 mice, 1 rabbit) using the tlood from Group A
animals. If parasite Count was high enough, a second passage would also be done, infecting (C]roup
A3: 10 mice) with Group A2 blood. The objective of these passages would be to ‘Select’ those
parasites that were most adapted to rodents.
Methods and Techniques
Infection. Infection of parasites was given intravenously to the goats and rabbits (the iri:ler
thigh being most successful for the rabbit) (Annex B. 1). The mice were injected intraperitonebllq
lt
was discovered that smaller needles (29 guage vs 18 guage) u ere needed when infecting baby rats.
as the larger guage needles incurred uncontrollable bleeding. When the source of parasites was t l\\c
goat, undilluted fiesh blood was injected to the new host. When infecting from the mouse or r-t, a
few drops of blood were taken fiom the base of the tail and dilluted in PSG before infecting into the
new host (Annex B.2). The level of parasiternia and condition of parasites being injected was always
checked (whether dilluted in PSG or not) before and afier infection.
Parsitemia Diagnosis. In most trypanosomiasis infectlons, parasiternia oscillates greatl;! due
to the endless supply of the parasite antigen coats. A whole parasitemia peak cari occur in two days
(ie one weekend) in which parasiternia Will go fiom one cross* to three cross and back to one cross,
thus requiring daily analysis of trypanosomal activity in the animals. There were two forms of
examination, direct and interphase.
12
Direct Examination: A single drop of blood was placed on a slide, protected with a caver slip, and
then examined under the microscope (magniILation 25X) for the presence of trypanosomes
The mice, wild rats, and rabbits were analyzed only through this direct examination of Ii-esh
blood. For the mice and rats blood was taken from the rip of the tail, using a blood lancet.
-: . .
Blood samples were taken from the rabbits and goats at the tip of the ear.
Inter-phase: When trypanosomes are centrifuged in fresh blood, they are concentrated at the
inter-phase between the white and red blood cells. Thus, to have a more sensitive anal-sis of
parasitemia, this second technique was used on the goats. A capillary tube of fresh blood was
taken fi-om tip of the ear and centrifùged in a microcentrifuge for three minutes ( 12,000
rev/sec). The tube was then tut just below the interphase, on the red blood ce11 side. Tl’c
inter-phase was then put onto a slide, covered with a caver slip, and then analyzed under the
microscope (Annex B.3,4). This method otien revealed parasitemia unfound in the fret;”
blood analysis.
“Parasiternia level was measured by the method described by Murray et a1 (ref) however the leYe1
was always taken fiom direct examination instead of interphase, thus making the parasiternia rn’~~:h
higher than calculated by Murray. According to Murray, one cross: I 02- 10”; two cross: 1 03-1 !Yi:
three cross: 5x1 03-5x1 04; four cross: 1 04-5x1 05; five cross: >5x i O5 (trypanosomes per ml) ”
Results and Interpretation
On the fourth day post infection (pi) 50% of the mice showed parasitemia on fresh blood
smears. Over the next seven days a11 but two mice were positive, with one mouse having rathe’ l’igh
parasiternia (three cross). The rabbit was positive days six through ten, wirh parasiternia the most
during the last two days of infection (Char-t 1). The duration and levd of the parasiternia was much
lower than expected and very little, if any, fluctuation was observed Because the first signs of
parasiternia came SO soon, the rodents seemed to be quite receptive hosts, thus the fnst passage was
postponed until a higher level of parasiternia was obtained I lnfortunately, rhe first peak was ;hc
only peak, making a serial passage impossible.
13
/
IRabbit 1 / Mice
:I .TLapl
~Il~~infect~~~~~--I~~.:~-
infect infect infect infect infe
1-1 -- -. .--11 .--!.-..---.i -.. .
!
/
0‘
0;
0;
0;
0:
0 0
0’
-2
Char-t 1
The results obtained were less than hoped for, however when ccmparing the prelimina~ t;
experiment’ with the follow-up work, one cari see a major dif‘frence which rnay ha\\.e been tht
determining factor. The mice in the preliminary experiment had been infected directbs with bloo!
from the sick Bambilor cattle The infection killed the cow. and the blood was unfortunately not
preserved in its original state The trypanosomes, however were alive and healthy for- a number oi’
months in the infected mice. In early December, 1997; blood iinm the infected mice was gi\\,en t;) ;I
Sahelian (trypano-susceptible) goat, CAP 2, in order to have a permanent stock of the strain i)ncc
the goat showed high parasitemia, blood was drawn and storeci at -80 C in licpid nitrogen
(parasitemia: three cross). When this blood was thawed in January, 1998: the parasiternia cc,unt was
rather low (one cross), SO in order to have higher parasiternia prier to rodent infection, the blood was
reinjected into the same goat (CAP 2).
Eighteen days afier infection, the goat’s parasiternia Count was at three cross, SO blood w:ts
drawn and injected into the Group A rodents (0.5 ml for each mouse, ! .O in the rabbit)
A
14
fluctuation of parasitemia was expected in the animals, as was seen in the preliminary experiment, SO
all the rodents were faithfully checked until 16 days of negative results caused a loss in faith of’ a
return of the parasitemia. By day 50 pi, five of the ten mice had died, assumedly fiom natural causes
as the last sign of trypanosomal infection was greater than or equal to 25 days before. Hiddtr:
4.:’ n
parasitemia (ie in the cerebral cavities) is not as common with T.vivax as with some species such as
T. brucei, thus when parasites were no longer in the fresh blood smears, the animals was consitlered
to have undergone spontaneous recovery.
One of the possible reasons for the discrepancy in results between the two experiments may
be found in the history of the Bambilor strain. The five mice that showed such high and enduring
parasiternia in September were infected directly with the blood from the Bambilor cow, which could
be considered as one passage. In the follow-up experiment however, that original source was
unavailable, but rather that which had been preserved in liquid ,+trogen. When counting the nimber
of passages the strain endured (including fieezing), it would be five: cow to mouse (1) to goat (2)
to liquid nitrogen (3), to goat (4) to Group A rodents (5). The impact of these numerous passages
on the transmissibility of the Bambilor strain is unclear. When this was reslized, returning to
Bambilor in hopes of finding a symptomatic host seemed to be most logical. However time and
transportation restraints removed the possibility.
Discussion
Although the parasiternia in the rodents was less than expected, it jhould not be neglected
One of the mice (F3) reached a peak of three cross on day 8 post infection, and three of the r lice had
detectible infections for six to seven days. During that week of infection, a rodent cari travel many
kilometers, coming into close contact with animals of numerous farms as well as other rodents.
Nevertheless, that infected rodent would be of little consequence unless there were a reasonable
means of transmitting the trypanosomes to other rodents or to larger farm animals. Tsetse flres do
15
not normally choose rodents as a source of food, thus it is perplexing that a ruminant form of
trypanosomiasis could be infective to rodents. It would seem necessary to have some sort of
exposure to rodents in order to undergo the needed adaptations.
In the Niayesregion, where the tsetse flie population is rather low, one is forced to consider
+y.’ 1.
the possibility of other mechanical vectors. Although the Stomoxyine and Tabinedea are more
numerous in the Niayes region than tsetse flies, they also do not normally feed on rodents. Crilien
extenuating circumstances however, the possibility cannot be ruled out. Secondary experimems
during the course of this internship revealed a high level of T.lewisi infection in wild rats, for M hich
“there is direct or inderect evidence that the intermediate hosts are fleas’1.7 Whether rodents’ fleas
feed on ruminants is not clearly documented but if the flea population is high, both rodents and farm
animals may be candidates for blood meals. The possibility of other species of fleas feeding on the
rodents must be considered, and it has been reported that both dog fleas (C’tenocephalides cu~lis)
and fleas of mari (Pulex irritans) cari be infected with ïIlewisi.7 Furthermore, the most recent
studies in lab have shown that the jrvivax strain is readily infective to wild rats. Studying the
feasibility of a flea being the mechanical vector for a 7:vivax strain would be very beneficial at this
time. In conclusion, although the results were not what were expected, the experiment caused
greater awareness in the many factors invovled in parasite transmissibility and host infection
Furthermore, the possibilty of the strain being infective to laboratory rodents (or even more
importantly, wild rats) is still not ruled out.
16
Secondary Projects
During the course of the internship there were a number of projects that were undertaken in addtion
to the main focus as described above. These projects were either the logical follow-up of laboratory
results, investigative observations, or exercises in learning a specific technique. Although they were
often without hypoth@:or strict objectives, they proved to be very instructional and benefkial.
Inducing trypano-tolerance
In mid December, 1997, the Sahelian goat CAP 2 was used to obtain a plentiful supply of the
Bambilor T.vivax strain. On the eighth day afier infection, a high level of parasitemia was found in
fresh blood smears. By day ten, parasiternia was at five cross and the goat was very symptomatic
As it appeared the goat would die within 24 heurs, it was treated with lO.Omg of Berenil and was
shortly thereafter clean of parasites.
A month and a half later, the same goat was infected with those same parasites--recover-ed
fi-om the frozen supply (3ml of blood, very feable parasiternia--one cross). The purpose of this
second injection was to obtain fresh, vivrant trypanosomes for injection into the rodents.
Nevertheless, as the parasiternia seemed to be oscillating and CAP 2 was showing little if any
symptoms, it was decided to leave the infection untreated to observe parasite behavior. Over tt:e
next 110 days, the parasitemia had over 17 peaks, ranging from five cross to no detectible
parasitemia (Annex A.2, solid line).
Mer 60 days, the parasitemia stayed at or below two cross, hplying that CAP 2 was i:nining
greater control over the infection. Such behavior is typical of trypano-tolerant breeds, SUC~ a:; the
N’Dama. Sahelian breeds are normally trypano-susceptible, as was observed in CAP 2 during the
first infection when it was naive. However, on re-introduction of those same parasites afler
treatmenl, the trypano-susceptible breed seemed to be rather tolerant. However, during the past four
months, CAP 2 has lost weight and started to show symptoms of fatigue due to maintaining c”
chronic disease, even though the parasite level is currently relatively low.
17
Because a normally trypano-susceptible goat had developed relative tolerance to the T.vivax
strain, a new naive Sahelian goat CAP 3 was obtained and infected with the Bambilor strain (2ml
fi-om freezer added to 2ml of fresh CAP 2 blood). AfIer five days parasitemia was evident in fresh
blood smears, and in the last 34 days there have been 7 peaks, with most of the parasitemia resting at
a high level of three ‘&8ss (Annex A.2, broken line). Not only is the parasitemia high, but CAP 3 is
also showing severe weight 10s~. The reliability of the daily hematocrit reading is not dependable, and
in neither of these goats has there been a systematic or conclusive change in hematocrit, which would
indicate the expected anemia.
When checking current literature, one finds similar trends as those discovered with these two
goats. In the early 1990’s the Leak group set out to study and control the tsetse population in cl
southwestern region of Ethiopia where there is a high prevalence of drug-resistant trypansomes.
They were able to reduce the trypanosomiasis cases and within their discussion, they hypothesized
that this apparent reduction in prevalence of trypanosomiasis cases was achieved
because under reduced tsetse challenge and the consequt:nt reduced re-infection rate,
diminazene aceturate became more effective in controlling parasitemia as cattle were
able to express signifïcant levels of acquired immunity”.’
When combining the field observations of the Leak group with our own laboratory results, it would
seem fair to suggest that trypano-susceptible breeds afier an initial treatment have a greater ability to
control their infection as compared to their naive counterpart. However one must be careful tc
qualify that with the condition that the re-infection rate is limited, reducing the number of challenges
the animal faces. Furthermore, when in their normal environment, trypanosomiasis infections are
normally transferred a.fIer undergoing a life cycle in the midgut of the tsetse fly. Not only did the
trypanosomes re-given to CAP 2 lack this, but they were exactly what the goat had dealt with less
than 2 months before. This sort of ‘identical mechanical transmission’ would scientifically seem to be
the best environment for building immunity and tolerance to the infection. Perhaps a reasonable
18
approach to trypanosomiasis control wouid be the infection-treatment method followed by vectol-
control, thus reducing the rate of re-infection, giving the animals an opportunity to acquire immunity.
Wild rats
There are m8ny known and unkown differences between labo;atory Balb C mice and the rats
that run between farrns in the Niayes region. Because of the possibility that the Bambilor 7: vi~czx
strain was infective to rodents, wild mice or rats were desirable for further investigation. Two rat
traps were set on Dr. Diaite’s farm in Niaga and within 48 hours afier setting traps, one male rat was
caught. Alter preliminary examinations, the rat was discovered to be infected with lr. Iewisi. l‘he
majority of T.lewisi strains are parisitic in rodents with only a few known exceptions.’ In order to
check the virulence of this strain in different animals, infected blood was given to a goat, three
female mice, and one rabbit. Alter over 30 days of caretil observation, none of the animals had any
detectible parasitemia, thus indicating that this 7:L~isi is indeed a rat-specific strain.
Three weeks afier the first rat was captured, another one was found, also infected wit5
Tlewisi. In hopes of using this one in hrther 1:vivax studies, it was desirable to remove the current
infection. Thus af?er five days of captivity, the rat was injecred with 1.5 mg of Beneril, suspended in
0.5 ml DI water, which proved to be fatal. It was assumed that either the drug is not meant for rats,
or more likely, that it was a toxic dose. Many African trypanosomiasis patients suffer from severe
venous damage alter undergoing chemotherapy for the infection. This problem led to experiments
testing various ways to administer anti-trypanosomal drugs. In 1996 Jennings reported that
melarsoprol is effective in treating mice infected with ï:bmcei hrwcei when put in20 a gel form and
applied topically instead of through the standard intravenous treatment.15 However, such treatment
of the wild rat in lab would have been next to impossible.
Over the course of one month, a total of three wild rats were caught, a11 showing high levels
of T.lewisi infection. Two of them were females and had pups shortly after arriving in captivity.
19
Because of the need to put the rats to sleep with ether in order to check parasiternia, daily
observations were abandoned afIer the babies were born. However, it was noteworthy that the baby
rats never showed parasiternia, although the mothers were positive during and afier pregnancy as
well as during nursing. This would confirm evidence that the T.lewisi infection is transferred within
Q‘ 2.
the rats by a vector, net via contact with an infected mother.
Separation of trypanosomes from blood
One of the most successful types of separation when ,isolating trypanosomes is ionic charge
separation (both simple filtration and the more complex chromotography). The system isolates
different items within a solution according to ionic charge (Figure 6). The trypanosomes correspond
to one particular peak, and cari thus be isolated in a number of tubes.
substance i-
peaks
according to L
charge
1 2 3 4 5 6 7
collection tubes
Figure 6
During the course of this intemship ionic charge separations were completed, using a DE52
chromotography slurry and PSG buffer (pH 8.0). Initially a standard buchner fIurne was used but it
was determined that a better separation could be achieved using a tut-off 15 ml polypropelene
centrifuge tube. Filter paper was put in the bottom to retain the slurry and a pipet tip was glued to
the end to aid in collection.
When setting up the column, the slurry must always be kept wet, although just prier to
adding the fresh blood the buffer level must be just at the top of the slurry. Blood was usually
dilluted 25%-50% with PSG buffer before running through the column. Once a11 the blood is in the
2 0
siurry, buffer is added to elute the trypanosomes. The DE52 siurry binds more tightly to the blood
cells than to the trypanosomes, SO one must be certain to begin checking for the parasites well before
blood cells are eluted. If the separation is good there are no blood cells in the collection tube, only
trypanosomes and PSÇ buffer. Mer separation, the collection cari be centrifuged and the pelle1
.q ‘f.
resuspended in PSG, but depending on the purpose of separation, this is ofien unnecessary.
When trypanosomes are in f+esh blood (in an heparinised tube), they have been known tc.
survive at room temperature (2530”Celcius) for over 48 hours (experience in lab). However, after
separation from blood and swimming in PSG alone, they are much more fragile and need to be kept
chilled at 4 C. Having the trypanosomes on ice immediately afier separation from the column is
crucial to the survival of the trypanosomes. Futhermore, T.vivax, which is particularly fragile, was
the only strain that was used for trypanosome separations during this internship as it was the oniy
species available in large quantities.
c10ning
The principle behind cloning trypanosomes is to isolate one single trypanosome and then
infect it into a host, with hopes that the single parasite Will multiply and create a uniform infection
within the host. TO increase chances of infection, immuno-comprimised animals are preferred, SUC~
as radiation treated rnice, or newborn animals. Because there was a shortage of laboratory miçe (let
alone newboms), the possibility of making an attempt at cloning was not hopefùl. However when the
wild rats were captured, two of which had babies shortly alter arriving in lab, it was considered to be
a good opportunity to try to clone trypanosomes.
When the second litter of pups was five days old, and goat CAP 3 had parasitemia of three
cross, an isolation (technique previously outlined) was completed and the trypanosomes put on ice
Because of the fragility of the T.vivax trypanosomes, and the need for a very healthy single parasite,
work was done quickly and conscientiously. The trypanosomes were diluted 10-100X, depending on
21
their concentration, and then one small drop of PSGltrypanosome solution was placed on an
indented slide and examined under the microscope. It was discovered that a higher concentration of
trypanosomes with a small drop of liquid was the most logical, as the parasites move SO quickly and
only in this manner could one be certain of the number of parasites on the slide. Theoretically, once
Y ’ .‘t.
a single parasite is isofated, plain PSG would then be added to the slide and then a11 the liquid drawn
into a syring and injected into the host.
However, the task proved to be incredibly difficult, and afier two days of attempting (and
failing) to isolate a single, healthy trypanosome the project was abandoned. The situation proved to
be rather frustrating because a number of times a single trypanosome was discovered on the skie but
by the time the whole slide was checked (which only took about 50 seconds), the parasite had died
This systamatic dying of parasites was attributed to the heat of the microscope lamp traumatizing
the parasites, both by the high temperature and the shock of the temperature change. If another
attempt at cloning were to be made, it would be suggested that a11 steps, especially microscopic
examinations, be done at the same temperature, preferably in a cold room.
22
Field Work
Dairy Farms, February 2 1
The Niayes r$gi;on is important for the agricultural and economic development of Senegal
The land has particularly favorable qualities for farming, and the closeness and easy transport of
goods to the export tenter of Dakar makes the development of this region strategic. Within the
Niayes there are two main types of dairy farms. The first are run by the traditional farmers who daily
take their cattle, goats, and sheep to graze on the open land. For generations these local animals
have had considerable exposure to the habitats of tsetse flies (and other biting flies) and ofien have at
least some, if not complete, resistance to trypanosomiasis. The second type of farm is newer and
usually a huge multi-million dollar organization that imports high-milk producing animals such as
Holsteins. Although these cattle show great promise for improving the milk and meat production of
the nation, the animals are usually naive to trypanosomiasis and particularly susceptible during t he
rainy season. Many of these mega farms keep their animals contained and have extensive insecticide
programs to reduce vector-born diseases. Nevertheless, the close proximity of the locally run fàrms
produces a frustrating situation for the mega-farm owners because the untreated, locally grazing
cattle bring home an assortment of diseases afier tramping through the valley. Regardless of the
strict containment of their animals, the large farms are directly affected by the health of the nearby
traditional dairy farts.
In mid-February an FAO officia1 who specializes in trypanosomiasis (Mr. Shizuka) came ao
assess the level of trypanosomiasis risk to dairy animals. Farms in the Niayes region were ofgreatest
concern, following the observation of the tsetse population in an animal park in Dakar.
Niacoulrab. The first farm which was in Niacoulrab, located just before Lac Rose, had 400
cattle (they had just recently reduced their population from :700.) The farm had a rather extensive
breeding program of local breeds with the larger and more milk-producing Holsteins (Annex 13 5).
23
The farm was well-equipped with milk machines, cold rooms, and spray stations. Most of their
cattle produce 30 L per day, with some as many as 36 L during the high season. Although the farm
does not have a serious problem with trypanosomiasis, they still administer prophylactic anti-
trypanosomal drugs to all the animals every 6 months. The greatest risk is during the rainy season,
*,k ’ .1.
.’
3uly-September, when food sources are low and the habitat is more condusive to vector-borne
diseases. Niacoulrab had recently bought some N’Dama cattle, and the veterinarian mentioned some
concern about them carrying trypanosomiasis infections (Annex B.6). This situation was indicative
of the fact that increased abilities to transport animals also enable the spread of otherwise isolated
diseases.
Wayembam. In the second farm we visited, Wayemham, a11 the cattle were Jerseys and had
been purchased from Denmark. They have 120 cattle, with the average production of 30 L per day.
The farm was much less-advanced than Niacoulrab and the cattle are located directly next to a major
route for the local grazing herds (Annex B.7). The veterinarian exprcssed concern for the
transmission of diseases from local animals, and mentioned that he had treated three animais for
trypanosomiasis. He also said that they were planning on building a new, completely enclosed
building for the peak-producing CO~S, as they were always more fragile. The farm’s milk is sold by
women in local kiosks at 500-600CFA, oRen as saur milk as it is not refrigerated (nor homogonized
or treated in any other fashion).
The last large dairy farm we visited was
SOCA.
SOCA, which is located in Sebikotane, very
near the herds in which the Bambilor T.vivax strain was found in September, 1997. SOCA is a
multi-million dollar organization that seemed to be in another class when compared to the two
previous farts. The veterinarian was very well-educated and had a clear concise presentation caf the
farms activities. He mentioned that their greatest problems with disease fa11 during/afIer the rainy
season when the food sources are limited. The soi1 in the area is very fertile, but water sources for
irrigation are difficult. The dairy cows are almost all Jerseys, with a couple other breeds--local and
24
imported. In order to boost their profits, they sel1 milk (raw and pasturized), cream, and juice
(bissap). Their two main customers are big distribution factories as well as the local kiosk and
boutiques. Nearby SOCA there was a strawberry field adjacent to a small area very conducive for a
tsetse fly gallery (Annex B.8).
ARer the dairy farms two other sites were visited as well, neither of which had animals but
were nonetheless interesting for the development of agriculture in the Niaye region. The first being
ENDA Syspro and the second a monastary, the Seminary of Libermann. The seminary had large
fruit tree orchards and had obviously been affected by recent years of drought. Regardless of the dry
climate, some of the monks were emphatic about the presence of tsetse flies on the seminary
grounds.
Park Bandia, March 30
In the beginning of February, 1998, an antelope in the Wild Animal Park Bandia (Annex
B.9,10) died due to a trypanosomiasis infection. Another animal was found dead in the Park in rhe
middle of March, but the cause of death could not be identified because of decomposition. In an
effort to assess the tsetse fly density within the par-k, nine fly traps were set on March 28th.
Two
days later we returned to retrieve the traps and their contents.
There were no tsetse flies or tabanides in any of the traps when we arrived. Of the 9 traps
set, 6 of them were placed along or very near the Somone creek and a11 of these had stomoxyines,
totalling 34. Although there was no bait put in the traps, the high Count of stomoxyines ( 13 in one of
the cages) indicated that the traps were successful and that the tsetse fly population is negligable, if
at ah. As mentioned before, Stomoxyitre are suspected mechanical vectors for trypanosomiasis, and
in an enclosed animal park where animais are more likely to be in close proximity to each other, the
success of mechanical transfer woufd seem to be more probable.
2 5
Sokone, April22-24
As this internship was completed during Senegal’s dry season, the Dakar region and Niayes
valley had very few, if any, tsetse flies. Thus, in an effort to observe a more suitable environment for
trypanosomiasis vectors, a trip to Sokone was planned. On April 22nd we left Dakar with 9 fly
a*:’ *
traps, three mice (Group C: Ml,MZ!,M3), and other supplies,
After arriving in Sokone we drove to the outskirts of the region, where two tsetse fly
galleries were located. The first site was Keur Aliou Gaye (KAG), a dry but classified forest located
adjacent to farming fields. It was divided into two sections, Ai in which we placed two traps (KAG
no 1, no 2) and about 3 kilometers down the road, Aii, where we placed two more (KAG no 3,4).
Both of these stops were very dry and had hard, dry soil. There was quite a bit of vegetation,
although not ver-y green. Aii had a huge monkey residency as well as domestic livestock
Alter driving through many villages we came to the second site, Foret Classe de Patako,
which was a much more moist and humid forest. The fohage was very thick and there were op~n
bodies of water used for irrigating the small plots of vegetables and trees. We placed five traps here
(FCP nol-no5), with much more hope of catching flies. At FCP 2 a fly landed on, and bit, Dr
Diaite’s hand and another one was seen on the back of his shirt (Annex B. 11). On our way out of
FCP, a tsetse fly entered the car and SO we captured and contained it until we arrived back in
Sokone, where it was discovered to be still alive, and a male Glossi~m morsitcms s~~bmorsittrrrs
The next morning we travelled down to the weekly market in Touba Nding, where there
would possibly be livestock infected with trypanosomiasis. We brought the three mice (GroL;p C) in
case a sick animal was found and the owner allowed us to draw blood. As we did not have access to
a microscope, all injections would be blind. Nevertheless, we took blood samples from three horses
and infected the mice. Group C Ml: 0.5 ml injection fiom adult male horse, slightly anemic with
swollen prescapular lymph nodes (Annex B. 12); owner--Modou Mbang, village--Passy Ndenderley.
Group C M2 1 .O ml injection from an adult male horse, slightly anemic with swollen lymph nodes
26
and hind legs; owner--Soulemane Dieng, village--Pakala. Group C M3 1 .O ml injected from a
Young female horse, slightly anemic but not very sick; owner--Moussa Sarr. In the afiernoon we left
for the tsetse fly sites which produced the following results:
Keur Aliou Gave
.
Foret Classe de Patako
,>‘ q.
IL4G 1: 9 Stomoxyines, appx 10 other flies
FCPI: lfly
KAG 2: 1 bee
FCP 2: 2 Glossine (1 m + 1 f), 2 Stomoxyines
KAG3: 1 wasp
FCP 3: 10 Glossine (5m + 5f)
KAG 4: 2 Stomoxyines, appx 4 other flies
FCP 4: appx 10 flies
FCP 5: 4 Glossine (3m -t lf), 5 Stomoxyines
When we arrived in Sokone, ah the flies were caref?rlly observed and rechecked. Al1 the tsetse fies
were identified as G.morsitans submorsitans as indicated by their “black sock” legs. Because KAG
was SO dry and unlikely to have tsetse flies, it was disregarded when calculating fly density. Thus
given the five traps at FCP, the overall density was calculated to be 3.2 flies per day, per trap
‘l‘his
fly density is relatively high, especially considering that it was during the dry season. Although
Glossinapalpalis were not found in any of the traps, it would be expected that they would be
present during the rainy season. G.palpalis need a more moist, humid climate and tend to follow
very closely to water ways. When the weather is drier, G.palpa1i.s Will retreat to pockets of humidity
and the G.morsitans Will retreat to the areas previously inhabitated by the G.palpalis.
If the chmate
is wet enough, G.morsitans Will abandon major water ways and spread out into the region. Such
migration habits affect not only the fly densities, but more importantly the two species’ host
selection.
27
fumex
28
P
0 ih
w
Kl in
1
A.
N
29
Annex
Parasiternia Level
--L in
--L
0
in
0
P b
P
cd 67
w
!Q m
Annex A.2
Parasiternia Level
rd
f
2 VI
2
0
u1
0
2y
5
8
11
14
17
20
23
26
29
32
35
38
41
44
47
68
71
74
77
80
83
86
89
92
95
98
101
104
107
n
Annex B. 1
Annex ES.2
31
Annex B.3
Annex B.4
32
Annex B.5
Annex B.6
33
. . . . .--
Annex B.7
Annex B.8
34
Annex Es.9
Annex B.10
35
Annex B. 1 Ii
Annex B.12
36
Bibliog-aphy
1) Balaban, N., Waithaka, H.K., Njogu, A.R., Goldman, R. Intraceliular Antigens (Microbutule-
Associated Protein Copurified with Glycosomal Enzymes)-Possible Vaccines against
Trypanosomiasis. The Journal of Infections Diseuses 172 (1995) 845-850.
y; ”
2) Baltz, T., Baltz, D., Giroud, C., Crockett, J., Cultivation in a semi-defined medium of animal
infective forms of Trypanosoma brzrcei, T. eqztiperdzrm, T evansi, 11 rhodesiense, and
Tgambiertse. ne lZkB0 Journal. 4 (1985) 1273-1277.
3) Diaite, A., et al. Work submitted Februarly 1998
4) Ekwanzala, M., Pepin, J., Khonde, N., Molisho, S., et al. In the heart ofdarkness: sleeping
sickness in Zaire. The Latzcet 348 (1996) 1427-l 430
5) Gouteux, J.P., Kounda Gboumbi, J.C., Noutoua, L., et al. Man-fly contact in the Gambian
trypanosomiasis focus of Nola-Biolo (Central African Republic). E-opical Medicine at~d
Parasitology. 44 (1993) 213-218
6) Hirumi, H. And Hirumi, K. Continuous Cultivation of ‘Ijl~~atzosoma brzrcei Blood Stream Forms
in a Medium Containing a Low Concentration of Serum Protein Without Feeder Cell Layers
ïhe Journal of Parasitology. 75 (1989) 985-990.
7) Hoare, Ceci1 A. The Trypanosomiasis of A4mr~rncr1.s
1972 Blackwell Scientific Publications
8) Kuzoe, F.A.S., Current situation of African trypanosomiasis Acta Copica 54 (1993) 153- 162
9) Leak, S.G.A., Peregrine, A.S., Mulatu, W., Rowlands, G.J., and D’leteren, G. Use of insecticied-
impregnated targets for the control of tsetse flies (G/o.wirm spp.) and trypanosomiasis
occurring in cattle in an area of south-west Ethiopia with a high prevalence of drug-resistant
trypanosomes. 5 (1996) 599-609.
10) Lumsden, Z.H.R. Trypanosomiasis. Zmmunokogy. 2 ( 1978) 1492- 1499.
11) McGovern, T.W., Williams, W., Fitzpatrick, J.E., Cetron, M.S., et al. Cutaneous manifestations
of Afiican trypanosomiasis. Arichives of Dermatology. 13 1 ( 1995) 117% 1182
12) Murray, M., Trail, J.C.M., Turner, D.A., and Wissocq, Y. l’roductivite Animale et
Tkypanotolerance.
1983, 9.
13) Winrock International. Assesment of Animal Agriculture itl ,Crrb-Saharan Africa. 1992
14) http://www.urmc.rochester.edu/smd/mbi/cann/224/Trypano.html
15) Jennings, F.W., Atougia, J.M., Murray, M. Topical chemotherapy for experimental murine
Alï-ican CNS-trypanosomiasis: the successful use of the arsenical, melarsoprol, combined
with the 5-nitroimidazoles, fexinidazole or MK-436. Tropical Medicine and lnternatiorral
HeaIth. 5 (1996) 590-598
3 7
A Study of Pertinant Regions
and Strains in Senegal
. -
by Crystal Waters
Intemship 1998
TSRA: Dakar, Senegal
Advisor: Dr. A. Diaite
Table of Contents
Introiluction
3
Literar y Review
5
The Trypanosome
The T\\:ector
Main 1,aborator-y Projet;
11
Introduction
Ol+ctives and Approach
Methods and Techniques
Results and Interpretations
Discussion
Seconuary Projects
1 7
Inducing trypano-tolerance
Wild rats
Separation of trypanosomes from hlood
Cloning
FielcI \\X’c,rk
23
Dairy Farrns in the Niayes
Par; Bandia
SotGMe
2 8
37
Introduction
“Trypanosomiasis of man is surpassed only by malaria as an infection that inhibits the
development of tropicallands”. l” It causes the suffering of ovl:r one million Africans (20,000 :I~:\\V
cases each year) and severe malnutrition due to the sickness and loss of livestock. i Trypanosomiasis
was first described in the fourteenth Century in what is IOW thc. country of* Mali. By 1902 two
English physicians in the Gambia ascribed the causative agent to be a parasite, and one year late;
another scientist identified the tsetse fly as the vector.” In 1906 Winston (‘hurchill stated that thc
population of Uganda had been reduced by sleeping sic,kness fi-om 6.5 to 2 5 million.4 The samc year
Ayres Kopke introduced Atoxyl, an arsenic compound for tre:ltment of thc disease, and by tht: carly
1950’s human sleeping sickness had been greatly minimized through surveillance programs and
vector control.‘4 However since the 1960’s trypanosomiasis has been on thf rise in Africa, as p:)st
independance social umest and lack of fùnds has causeCI many control and tleatment programs t. be
neglected..“8 Today sleeping sickness occurs in 36 Afrlcan coi.ntries in which more than 50 million
people are at risk.
Animal sickness and mortality due to trypanosomal infections ha\\,e placed even greater
constraints on development in tropical lands. Anemic animals nnpedt: agriculture development atld
contribute to severe malnutrition There are fifty times as many pathogenic jpecies that affect
animals than do humans, and some of these are transmitted in itreas uninfected by traditional vectors,
for example the tsetse fly. In Sub-Saharan Africa the annual loss due to animal mortality and
sickness was reported in 1991 to be approximately (US) $4 billion, representing 24% of the total
livestock production.‘3
This is particularly detrimental as the continent’s population is increasing hy
3.1% each year and severe malnutrition calls for an inc; ease, net decrease, of animal production ln
an effort to improve public health, the World Bank set .i goal for the continent of Africa of an aIrrua
food production growth rate of 4% by the year 2025.i7
This will only be possible with a containment
3
of the critical animal diseases. Trypanosomiasis is a particularly important disease as animals cari
produce partial resistance for several months to years, tiuring which time the animal is alive but
anemic and often unable to procreate or have good productivity levels.
Senegal is a country with variety in climate, ranging from the arid north to the sub-hum4
-,: *
south and south-east. Much of the state would be considered semi-arid, in which low rain fa11 is
particularly suitable for animal raising. The western Niayes valley just north of Dakar is well knolvn
for it’s potential as a very profitable farming region. Tire area \\\\;as infested with tsetse flies until t11e
early 1970’s when a control campaign was implementeti This was followeti up in the 1980’s with a
second phase which included insecticide impregnated traps as well as the prcvious method of
ground-spraying.’ However since mid 1997 tsetse flies have been reported in various parts of thc
region and every rainy season farmers in the Niayes report cases of trypanosmiasis in their herds
(most often 7:vi~ux). Yet whether tsetse flies alone or other biting flies as well are responsible is not
clear as no fly serveys are being carried out. Most of tht: technologically advanced farms havf:
implemented seasonal animal-spraying programs to reduce the incidence oi‘vector-born disease:;
Considering the importance of the Niayes regicn in Senegal, nejv surveillance and contrc:l
programs are being implemented by organizations such as FAO. The main project completed w Ilris
intemship was using a strain of trypanosomiasis recently recovered from a symptomatic cow in
Bambilor; located in the Niayes valley. The focus of the study was on the virulence of this strain in a
variety of hosts, ruminants and rodents. Other smaller projects were completed during the courst’ of
the internship and are included in the report as they adlled to the comprehension of the study
A I~W
field visits were also made to trypanosomiasis-infested areas in Senegal for observation oftsetse-host
interaction in the natural environment.
4
Literary Review
The Trypanosome
The trypanosome is a protazoan parasite in the class mastigophora As of 1972 there w:xe
over one hundred subgenus identified, classified in two main groups: Stercfx-aria and Salivaria
(infection via feces or saliva, respectively). The subgenus in Stercoraria are far more numerou;,
although those in Salivaria are often more pathogenic (Figure 1). The trypanosorne’s physical
morphology varies greatly among subgenus, although the basic structure is univcrsal (Figure 2)
kinetoplast.
A”
‘\\
undulating membrane
flagellum
(Figure 2)
The largest trypanosomes are found in the subgenus kfegatrypamrm and cari be as long as 13Oum
(T.ilzyerzs) and 15um wide (1Itheileri), whereas the smaller Nutmomorma.s
crin be as short as 8c~r11 iu
1ength.7 The body surface of the trypanosome is a fairly strong membrane, known as the pelhcle OI
periplast, which consists of three layers. Underneath the pellicle lies a network of nearly 100
microtubles which help to maintain the shape of the parasite. The parasite has a single flagellunr
originating in the kinetoplast, enabling it to swim freely within the host’s bloodstream. The lengt 11 of
the flagellum varies greatly among the different subgenus of trypanosomes. The use of biornetl: a>
well as the behavioral pattems in fresh blood cari help in identifying the subgenus of a parasite.
Some are very quick and traverse a microscope field with incredible speed (7:vivcrx) whereas otlters
appear very lethargic and even motionless at times (ïIJzris).7 Three different species were studied in
lab during the course of the internship: 1: vivax, 7: coppIet~.~e, 7: lewisi.
5
be uninfective to rodents but has been found in ungulates such as sheep, goats, and camels with
bovines being the principle host7 Pathogenicity varies, from Nigerian Zebu cattle who have an
untreated mortality rate of 100% at?er 3-4 months of exposure to some cattle in the Congo which
have been known to spontaneously recover alter 2 months of a Tvivax infection. Both wild and
.,1 _:
domestic ruminants are susceptible, with periods of incubation varying from 4-2 1 days for virulent
strains and up to 59 days for mild cases. Parasiternia ?:vels fluctuate g-eatly, even within one day,
although parasites are often more numerous in the first few months of infectlon
i?congolense is in the subgenus Nannomonas, often lacks a free flagellum, and is usua!l~,
smaller (8-24um) than Tvivax (Figure 3b) ‘/:cor~~o/t?~~ may be one of thc most econmically
important species of trypanosomes because “a11 species of domestic mammals are susceptible to
infection”.7p445 Within the many subspecies of congolense there is an apparant trend of greater
pathogenicity with the longer forms. Although 1:congolerrse kills most hosts within 6- 12 weeks
afier infection, there are reports of spontaneous recovery. Laboratory animais (including rodents)
are easily infected, although occasional strains require ser-ial passages from the original natural host
Figure 3
Host resistance to both Tvivax and ï:cmplerrse infection is greatly improved by good
nutrition and sanitary conditions. The N’Dama cattle (an indigenous breed ti, Africa) are weli krlown
to be tolerant to both Tvivax and Kcorrgolensc yet immunity may be an acquired trait, as cattle with
extended unexposure show greater susceptibility.7 On the other hand, Zebu cattle (originating in
Asia) show high levels of susceptibility regardless of exposure to trypanosomiasis.
The third species studied in lab is ?Yervisi which is in the fecal transmitted group of
Stercoraria. The average length of Tlewisi is 26-34um and on.3 of the distinguishing factors is a
7
(Subgenus) Megatrypanum
T. ingens (ruminants)
T.p(fami (bats)
. ..32
Hupe tosoma
T. lewisi (rats)
..‘; _.
T.prinmluni
Group Stercoraria
T. rarigeli
. ..53
1 Schizotrypcnmm
7: cr~zi jman)
T. hipposideri
. . 9
Genre Trypanosoma
7: viwx [ruminants)
Group Salivaria
II(7 :) b. brucri
1: (‘I~)b.gamhicil.sc
(mari)
densierlsct (~11s n)
Figure 1
T.vivux is in the group Salivaria, subgenus Duttonella and its size cari range from 18-3 1 )II~I
(including flagellum) with the majority being 20-26pm.’ The distinctively large kinetoplast (diameter
1.1 pm) and long free flowing flagellum help when identifiing 7: vivax (Figure 3a). Multiplication IS
via binary division and is initiated at the kinetoplast. 7: vivax is “xe of the most important and
widespread forms of trypanosomiasis of livestock in tropical Africa”.7 p “’ The strain is believed lo
6
rather long, rigid, and pointy posterior end; whose distance from the kinetoplast is longer than found
in 7:congolense or T.vivax (Figure 4). The principle (and perhaps only) host of Tlewisi is rats, most
commonly in Black and Brown rats which are found all over the globe.7 Incubation is approximately
6 days, after which the parsite undergoes rapid multiplication until the host’s immunity responds,
killing many of the parasites, leaving only adult forms in the blood. In the majority of cases, ~:~WV.YI
is nonpathogenic although pathogenicity has been attained through rapid serial passages7 In 193 3
one case was reported in a Malian Child whose home \\I’as infested with infected rats7
Figure 4
Although the trypanosome is one of the most studied l)rc)tzo;m parasites, it has eluded the
scientific development of an effective, long term vaccine. As Salivaria species are the most
pathogenic, study has been focused on the few species that are found within that group. One of the
more difficult factors is that the parasite evades the immune response by changing its external antigen
coat every few days. Just as a host has begun to identify and fïght the infèction, the parasite changes
outfits and runs rampant again. l” This scenerio produces peaks of parasitemia, much like the
oscillating temperature of a malaria patient. Trypanosomiasis is also dificult to study because the
parasite is rather fragile outside of a live host environmznt. In the late 1980’s scientists were st.ili
working on suitable growth medias which do not require feeder-cells for the infective form of Lhe
parasite.z6 Earlier studies claimed that the crucial ingredient was cysteine whereas the Baltz and
Hirumi lab groups have found animal serum and reducing agents (thioglycerol or 2-mercaptoethanol)
to be the determining growth factors.2’6
8
As well as the detailed studies mentioned above, more fundamental and observational
research is being done on control methods and vector-host relationships. The late diagnosis of‘an
American safari tourist (9 weeks afier exposure) with Ithodesizn trypanosomiasis reveals the
dificulty of identifying the disease, and extent of unawareness in the United States.” In the earlq
i’. . .*’
1990’s in Ethiopia, the Leak group found that insecticide-impregnated targets were effective for the
control of tsetse flies and trypanosmiasis in cattle herds.(’ While observing the behavioral patterns of
vector-host interactions in the Central African Republic (tsetse flies with pigs and humans), it 1~2s
concluded that a low densitiy of flies led to a greater proportion of human blood meals. ’
The Vector
The cyclical vector of Salivarian trypanosomes is the tsetse fly (~lossirrn. Glossines are long,
brownish black flies ranging 6-16 mm in length and easily identified by two distincive characteri:;tics,
crossed wings when sitting at rest and the cellule discale in the extended wing which is hatchet
shaped instead of the normal triangle (figure S(a)).
(a) cellule discale
Al1 30 species and subspecies of Glossina are present only in Sub-Saharan Africa, two of which are
found in Senegal: Glossina palpalis palpalis and Glossina morsitans submorsitans. G.pa1pali.s and
G.morsitnans are distinguishable most easily by their leg coloring. On the C;.pdpdi.s a11 five sections
on the leg are black (stockings), whereas only the last two on G.mar.sitcm.s (socks) (Figure 5(b)lI
9
Tsetse flics do not require blood meals every day, although on an empty stomach a fly cari
take up to its own weight in blood (155 mg) within 25 seconds. At the beginning of a blood mcal
saliva is deposited into the host to reduce blood coagu!ation and it is during this time that a great
quantity of trypanosomes cari be transmitted. One of the most scientifically important factors of thc
‘i .,
tsetse is their unique reproductive systems. Similar to the flesh-eating screwworms, female tsetse
flics are only fertile at the beginning of their lives because the reproductive anatomy is such that afier
an initial mating no more sperm cari enter. During the life of the fly, semen is delivered into the
uterus for fertalization. This one-time mating reproductive system has led scientists to develo;,
control programs using sterile males. Unfortunately, releasing sterile male tsetse flies has its dra\\~
back as they temporarily increase the possibility of disease transmission (unlike screwworms because
the male is harmless).
There is evidence that some species of Salivarian trypanosomixis cari also be transmitte+
mechanically by other biting flies, such as Stomoxyine and ‘làhirlidea. Unlike with Glossincr, u.:~icll
play a developmental role in the life cycle of the parasite, mechanical transfer is more like a
laboratory infection with a syringe. Such transmission must be quick (3-5 min), as success depeqds
on the survival of the parasites on the mouth parts of the fly. 7irbinide.s are particularly menacinS. as
their saliva has an anticuagulate that causes hemoraging at the site of bite, yet unlike Glossir~a and
Stomoxyine, only the females are hematophages (blood eaters).
The last species of trypanosome studied in lab was T.lewisi, which is cyclically transmittcd by
the rat-flea, Xenopylla cheopis. However, “a number of observers have demonstrated that this
trypanosome cari also develop in other fleas’17 p227 such as those that attack dogs, mice, or hur;ans
The development cycle in the tlea is about 5 days, aRer which they are discharged in the feces of the
flea, infecting a new host at the site of the bite
10
Main Laboratoxy Proj ect
Introduction
In early Septe@.er, 1997 a small farm owner in the Bambilor region complained of animal
sickness and death in cattle and goats. Blood from one of these animals was taken to Dakar where
laboratory tests (fresh blood smears) revealed a well progressed 7Ivivax infection. Prior to
analyzation, five mice were injected with the infected blood. A few days later the death of one I )t
the mice led to the examination of the remaining four, one of which showed positive parasitemia
The infection was monitored daily and fluctuating parasiternia was observed in one of these mtct’ f’~,l
over one hundred days (Annex A. 1). A single passage given to five other mice (two naive) produced
parasitemia in four, the longest of which lasted over sixty days.’
T. (‘ttorzella) vivax was first identified by Ziemann ( 1905) in domestic ruminants and ovtx
the past Century a vast number of T.vivax strains have been discovered, none of which “was nclrmally
infective to rodents.” 7 @03 If the species is indeed isolated to ruminants, then epidemics cari be
avoided by quick treatment of symptomatic animals. This has been very helpful in the Niayes regiol:
of Senegal, where T.vivax is known to be a problem. As mentioned previously, there is evide,lce that
Zvivax cari be mechanically transmitted by biting flies other f.han Glossina. The possibility that a
strain is transimissable to un-immunosuppressed rodents poses a threat to containment programs
The parasitemia observed in the fïve mice directly infected from the Bambilor cattle wus
notable in three ways: long duration, high level of para:;itemia, and non-pathogenicity. All of’ thesc
factors contribute to making rodents very good intermediary hosts, both between farms and also as
an uncontainable animal reservoir during non-endemic periods (ie Senegal’s dry season). As fiir:her
investigation of the Bambilor strain was needed, this internship was the follow-up experiment with
the following hypothesis: in the Niayes region, a strain of ‘/: (I.hrtto~rella) vivax is transmissible and
infective to laboratory rodents.
II
Objectives and Approach
The objective of this project was to confirm the results of the previous experiments (which
showed high levels of parasiternia in 83% of the infected rodents) With a larger group of animais
than previously used (Group A: 10 mice, 1 rabbit), the rodents would be infected with the Bambilor
T. vivax strain and daily observed for parasiternia. When the parasite Count was high enough, the
first serial passage would be done, (group A2: 10 mice, 1 rabbit) using the tlood from Group A
animals. If parasite Count was high enough, a second passage would also be done, infecting (C]roup
A3: 10 mice) with Group A2 blood. The objective of these passages would be to ‘Select’ those
parasites that were most adapted to rodents.
Methods and Techniques
Infection. Infection of parasites was given intravenously to the goats and rabbits (the iri:ler
thigh being most successful for the rabbit) (Annex B. 1). The mice were injected intraperitonebllq
lt
was discovered that smaller needles (29 guage vs 18 guage) u ere needed when infecting baby rats.
as the larger guage needles incurred uncontrollable bleeding. When the source of parasites was t l\\c
goat, undilluted fiesh blood was injected to the new host. When infecting from the mouse or r-t, a
few drops of blood were taken fiom the base of the tail and dilluted in PSG before infecting into the
new host (Annex B.2). The level of parasiternia and condition of parasites being injected was always
checked (whether dilluted in PSG or not) before and afier infection.
Parsitemia Diagnosis. In most trypanosomiasis infectlons, parasiternia oscillates greatl;! due
to the endless supply of the parasite antigen coats. A whole parasitemia peak cari occur in two days
(ie one weekend) in which parasiternia Will go fiom one cross* to three cross and back to one cross,
thus requiring daily analysis of trypanosomal activity in the animals. There were two forms of
examination, direct and interphase.
12
Direct Examination: A single drop of blood was placed on a slide, protected with a caver slip, and
then examined under the microscope (magniILation 25X) for the presence of trypanosomes
The mice, wild rats, and rabbits were analyzed only through this direct examination of Ii-esh
blood. For the mice and rats blood was taken from the rip of the tail, using a blood lancet.
-: . .
Blood samples were taken from the rabbits and goats at the tip of the ear.
Inter-phase: When trypanosomes are centrifuged in fresh blood, they are concentrated at the
inter-phase between the white and red blood cells. Thus, to have a more sensitive anal-sis of
parasitemia, this second technique was used on the goats. A capillary tube of fresh blood was
taken fi-om tip of the ear and centrifùged in a microcentrifuge for three minutes ( 12,000
rev/sec). The tube was then tut just below the interphase, on the red blood ce11 side. Tl’c
inter-phase was then put onto a slide, covered with a caver slip, and then analyzed under the
microscope (Annex B.3,4). This method otien revealed parasitemia unfound in the fret;”
blood analysis.
“Parasiternia level was measured by the method described by Murray et a1 (ref) however the leYe1
was always taken fiom direct examination instead of interphase, thus making the parasiternia rn’~~:h
higher than calculated by Murray. According to Murray, one cross: I 02- 10”; two cross: 1 03-1 !Yi:
three cross: 5x1 03-5x1 04; four cross: 1 04-5x1 05; five cross: >5x i O5 (trypanosomes per ml) ”
Results and Interpretation
On the fourth day post infection (pi) 50% of the mice showed parasitemia on fresh blood
smears. Over the next seven days a11 but two mice were positive, with one mouse having rathe’ l’igh
parasiternia (three cross). The rabbit was positive days six through ten, wirh parasiternia the most
during the last two days of infection (Char-t 1). The duration and levd of the parasiternia was much
lower than expected and very little, if any, fluctuation was observed Because the first signs of
parasiternia came SO soon, the rodents seemed to be quite receptive hosts, thus the fnst passage was
postponed until a higher level of parasiternia was obtained I lnfortunately, rhe first peak was ;hc
only peak, making a serial passage impossible.
13
/
IRabbit 1 / Mice
:I .TLapl
~Il~~infect~~~~~--I~~.:~-
infect infect infect infect infe
1-1 -- -. .--11 .--!.-..---.i -.. .
!
/
0‘
0;
0;
0;
0:
0 0
0’
-2
Char-t 1
The results obtained were less than hoped for, however when ccmparing the prelimina~ t;
experiment’ with the follow-up work, one cari see a major dif‘frence which rnay ha\\.e been tht
determining factor. The mice in the preliminary experiment had been infected directbs with bloo!
from the sick Bambilor cattle The infection killed the cow. and the blood was unfortunately not
preserved in its original state The trypanosomes, however were alive and healthy for- a number oi’
months in the infected mice. In early December, 1997; blood iinm the infected mice was gi\\,en t;) ;I
Sahelian (trypano-susceptible) goat, CAP 2, in order to have a permanent stock of the strain i)ncc
the goat showed high parasitemia, blood was drawn and storeci at -80 C in licpid nitrogen
(parasitemia: three cross). When this blood was thawed in January, 1998: the parasiternia cc,unt was
rather low (one cross), SO in order to have higher parasiternia prier to rodent infection, the blood was
reinjected into the same goat (CAP 2).
Eighteen days afier infection, the goat’s parasiternia Count was at three cross, SO blood w:ts
drawn and injected into the Group A rodents (0.5 ml for each mouse, ! .O in the rabbit)
A
14
fluctuation of parasitemia was expected in the animals, as was seen in the preliminary experiment, SO
all the rodents were faithfully checked until 16 days of negative results caused a loss in faith of’ a
return of the parasitemia. By day 50 pi, five of the ten mice had died, assumedly fiom natural causes
as the last sign of trypanosomal infection was greater than or equal to 25 days before. Hiddtr:
4.:’ n
parasitemia (ie in the cerebral cavities) is not as common with T.vivax as with some species such as
T. brucei, thus when parasites were no longer in the fresh blood smears, the animals was consitlered
to have undergone spontaneous recovery.
One of the possible reasons for the discrepancy in results between the two experiments may
be found in the history of the Bambilor strain. The five mice that showed such high and enduring
parasiternia in September were infected directly with the blood from the Bambilor cow, which could
be considered as one passage. In the follow-up experiment however, that original source was
unavailable, but rather that which had been preserved in liquid ,+trogen. When counting the nimber
of passages the strain endured (including fieezing), it would be five: cow to mouse (1) to goat (2)
to liquid nitrogen (3), to goat (4) to Group A rodents (5). The impact of these numerous passages
on the transmissibility of the Bambilor strain is unclear. When this was reslized, returning to
Bambilor in hopes of finding a symptomatic host seemed to be most logical. However time and
transportation restraints removed the possibility.
Discussion
Although the parasiternia in the rodents was less than expected, it jhould not be neglected
One of the mice (F3) reached a peak of three cross on day 8 post infection, and three of the r lice had
detectible infections for six to seven days. During that week of infection, a rodent cari travel many
kilometers, coming into close contact with animals of numerous farms as well as other rodents.
Nevertheless, that infected rodent would be of little consequence unless there were a reasonable
means of transmitting the trypanosomes to other rodents or to larger farm animals. Tsetse flres do
15
not normally choose rodents as a source of food, thus it is perplexing that a ruminant form of
trypanosomiasis could be infective to rodents. It would seem necessary to have some sort of
exposure to rodents in order to undergo the needed adaptations.
In the Niayesregion, where the tsetse flie population is rather low, one is forced to consider
+y.’ 1.
the possibility of other mechanical vectors. Although the Stomoxyine and Tabinedea are more
numerous in the Niayes region than tsetse flies, they also do not normally feed on rodents. Crilien
extenuating circumstances however, the possibility cannot be ruled out. Secondary experimems
during the course of this internship revealed a high level of T.lewisi infection in wild rats, for M hich
“there is direct or inderect evidence that the intermediate hosts are fleas’1.7 Whether rodents’ fleas
feed on ruminants is not clearly documented but if the flea population is high, both rodents and farm
animals may be candidates for blood meals. The possibility of other species of fleas feeding on the
rodents must be considered, and it has been reported that both dog fleas (C’tenocephalides cu~lis)
and fleas of mari (Pulex irritans) cari be infected with ïIlewisi.7 Furthermore, the most recent
studies in lab have shown that the jrvivax strain is readily infective to wild rats. Studying the
feasibility of a flea being the mechanical vector for a 7:vivax strain would be very beneficial at this
time. In conclusion, although the results were not what were expected, the experiment caused
greater awareness in the many factors invovled in parasite transmissibility and host infection
Furthermore, the possibilty of the strain being infective to laboratory rodents (or even more
importantly, wild rats) is still not ruled out.
16
Secondary Projects
During the course of the internship there were a number of projects that were undertaken in addtion
to the main focus as described above. These projects were either the logical follow-up of laboratory
results, investigative observations, or exercises in learning a specific technique. Although they were
often without hypoth@:or strict objectives, they proved to be very instructional and benefkial.
Inducing trypano-tolerance
In mid December, 1997, the Sahelian goat CAP 2 was used to obtain a plentiful supply of the
Bambilor T.vivax strain. On the eighth day afier infection, a high level of parasitemia was found in
fresh blood smears. By day ten, parasiternia was at five cross and the goat was very symptomatic
As it appeared the goat would die within 24 heurs, it was treated with lO.Omg of Berenil and was
shortly thereafter clean of parasites.
A month and a half later, the same goat was infected with those same parasites--recover-ed
fi-om the frozen supply (3ml of blood, very feable parasiternia--one cross). The purpose of this
second injection was to obtain fresh, vivrant trypanosomes for injection into the rodents.
Nevertheless, as the parasiternia seemed to be oscillating and CAP 2 was showing little if any
symptoms, it was decided to leave the infection untreated to observe parasite behavior. Over tt:e
next 110 days, the parasitemia had over 17 peaks, ranging from five cross to no detectible
parasitemia (Annex A.2, solid line).
Mer 60 days, the parasitemia stayed at or below two cross, hplying that CAP 2 was i:nining
greater control over the infection. Such behavior is typical of trypano-tolerant breeds, SUC~ a:; the
N’Dama. Sahelian breeds are normally trypano-susceptible, as was observed in CAP 2 during the
first infection when it was naive. However, on re-introduction of those same parasites afler
treatmenl, the trypano-susceptible breed seemed to be rather tolerant. However, during the past four
months, CAP 2 has lost weight and started to show symptoms of fatigue due to maintaining c”
chronic disease, even though the parasite level is currently relatively low.
17
Because a normally trypano-susceptible goat had developed relative tolerance to the T.vivax
strain, a new naive Sahelian goat CAP 3 was obtained and infected with the Bambilor strain (2ml
fi-om freezer added to 2ml of fresh CAP 2 blood). AfIer five days parasitemia was evident in fresh
blood smears, and in the last 34 days there have been 7 peaks, with most of the parasitemia resting at
a high level of three ‘&8ss (Annex A.2, broken line). Not only is the parasitemia high, but CAP 3 is
also showing severe weight 10s~. The reliability of the daily hematocrit reading is not dependable, and
in neither of these goats has there been a systematic or conclusive change in hematocrit, which would
indicate the expected anemia.
When checking current literature, one finds similar trends as those discovered with these two
goats. In the early 1990’s the Leak group set out to study and control the tsetse population in cl
southwestern region of Ethiopia where there is a high prevalence of drug-resistant trypansomes.
They were able to reduce the trypanosomiasis cases and within their discussion, they hypothesized
that this apparent reduction in prevalence of trypanosomiasis cases was achieved
because under reduced tsetse challenge and the consequt:nt reduced re-infection rate,
diminazene aceturate became more effective in controlling parasitemia as cattle were
able to express signifïcant levels of acquired immunity”.’
When combining the field observations of the Leak group with our own laboratory results, it would
seem fair to suggest that trypano-susceptible breeds afier an initial treatment have a greater ability to
control their infection as compared to their naive counterpart. However one must be careful tc
qualify that with the condition that the re-infection rate is limited, reducing the number of challenges
the animal faces. Furthermore, when in their normal environment, trypanosomiasis infections are
normally transferred a.fIer undergoing a life cycle in the midgut of the tsetse fly. Not only did the
trypanosomes re-given to CAP 2 lack this, but they were exactly what the goat had dealt with less
than 2 months before. This sort of ‘identical mechanical transmission’ would scientifically seem to be
the best environment for building immunity and tolerance to the infection. Perhaps a reasonable
18
approach to trypanosomiasis control wouid be the infection-treatment method followed by vectol-
control, thus reducing the rate of re-infection, giving the animals an opportunity to acquire immunity.
Wild rats
There are m8ny known and unkown differences between labo;atory Balb C mice and the rats
that run between farrns in the Niayes region. Because of the possibility that the Bambilor 7: vi~czx
strain was infective to rodents, wild mice or rats were desirable for further investigation. Two rat
traps were set on Dr. Diaite’s farm in Niaga and within 48 hours afier setting traps, one male rat was
caught. Alter preliminary examinations, the rat was discovered to be infected with lr. Iewisi. l‘he
majority of T.lewisi strains are parisitic in rodents with only a few known exceptions.’ In order to
check the virulence of this strain in different animals, infected blood was given to a goat, three
female mice, and one rabbit. Alter over 30 days of caretil observation, none of the animals had any
detectible parasitemia, thus indicating that this 7:L~isi is indeed a rat-specific strain.
Three weeks afier the first rat was captured, another one was found, also infected wit5
Tlewisi. In hopes of using this one in hrther 1:vivax studies, it was desirable to remove the current
infection. Thus af?er five days of captivity, the rat was injecred with 1.5 mg of Beneril, suspended in
0.5 ml DI water, which proved to be fatal. It was assumed that either the drug is not meant for rats,
or more likely, that it was a toxic dose. Many African trypanosomiasis patients suffer from severe
venous damage alter undergoing chemotherapy for the infection. This problem led to experiments
testing various ways to administer anti-trypanosomal drugs. In 1996 Jennings reported that
melarsoprol is effective in treating mice infected with ï:bmcei hrwcei when put in20 a gel form and
applied topically instead of through the standard intravenous treatment.15 However, such treatment
of the wild rat in lab would have been next to impossible.
Over the course of one month, a total of three wild rats were caught, a11 showing high levels
of T.lewisi infection. Two of them were females and had pups shortly after arriving in captivity.
19
Because of the need to put the rats to sleep with ether in order to check parasiternia, daily
observations were abandoned afIer the babies were born. However, it was noteworthy that the baby
rats never showed parasiternia, although the mothers were positive during and afier pregnancy as
well as during nursing. This would confirm evidence that the T.lewisi infection is transferred within
Q‘ 2.
the rats by a vector, net via contact with an infected mother.
Separation of trypanosomes from blood
One of the most successful types of separation when ,isolating trypanosomes is ionic charge
separation (both simple filtration and the more complex chromotography). The system isolates
different items within a solution according to ionic charge (Figure 6). The trypanosomes correspond
to one particular peak, and cari thus be isolated in a number of tubes.
substance i-
peaks
according to L
charge
1 2 3 4 5 6 7
collection tubes
Figure 6
During the course of this intemship ionic charge separations were completed, using a DE52
chromotography slurry and PSG buffer (pH 8.0). Initially a standard buchner fIurne was used but it
was determined that a better separation could be achieved using a tut-off 15 ml polypropelene
centrifuge tube. Filter paper was put in the bottom to retain the slurry and a pipet tip was glued to
the end to aid in collection.
When setting up the column, the slurry must always be kept wet, although just prier to
adding the fresh blood the buffer level must be just at the top of the slurry. Blood was usually
dilluted 25%-50% with PSG buffer before running through the column. Once a11 the blood is in the
2 0
siurry, buffer is added to elute the trypanosomes. The DE52 siurry binds more tightly to the blood
cells than to the trypanosomes, SO one must be certain to begin checking for the parasites well before
blood cells are eluted. If the separation is good there are no blood cells in the collection tube, only
trypanosomes and PSÇ buffer. Mer separation, the collection cari be centrifuged and the pelle1
.q ‘f.
resuspended in PSG, but depending on the purpose of separation, this is ofien unnecessary.
When trypanosomes are in f+esh blood (in an heparinised tube), they have been known tc.
survive at room temperature (2530”Celcius) for over 48 hours (experience in lab). However, after
separation from blood and swimming in PSG alone, they are much more fragile and need to be kept
chilled at 4 C. Having the trypanosomes on ice immediately afier separation from the column is
crucial to the survival of the trypanosomes. Futhermore, T.vivax, which is particularly fragile, was
the only strain that was used for trypanosome separations during this internship as it was the oniy
species available in large quantities.
c10ning
The principle behind cloning trypanosomes is to isolate one single trypanosome and then
infect it into a host, with hopes that the single parasite Will multiply and create a uniform infection
within the host. TO increase chances of infection, immuno-comprimised animals are preferred, SUC~
as radiation treated rnice, or newborn animals. Because there was a shortage of laboratory miçe (let
alone newboms), the possibility of making an attempt at cloning was not hopefùl. However when the
wild rats were captured, two of which had babies shortly alter arriving in lab, it was considered to be
a good opportunity to try to clone trypanosomes.
When the second litter of pups was five days old, and goat CAP 3 had parasitemia of three
cross, an isolation (technique previously outlined) was completed and the trypanosomes put on ice
Because of the fragility of the T.vivax trypanosomes, and the need for a very healthy single parasite,
work was done quickly and conscientiously. The trypanosomes were diluted 10-100X, depending on
21
their concentration, and then one small drop of PSGltrypanosome solution was placed on an
indented slide and examined under the microscope. It was discovered that a higher concentration of
trypanosomes with a small drop of liquid was the most logical, as the parasites move SO quickly and
only in this manner could one be certain of the number of parasites on the slide. Theoretically, once
Y ’ .‘t.
a single parasite is isofated, plain PSG would then be added to the slide and then a11 the liquid drawn
into a syring and injected into the host.
However, the task proved to be incredibly difficult, and afier two days of attempting (and
failing) to isolate a single, healthy trypanosome the project was abandoned. The situation proved to
be rather frustrating because a number of times a single trypanosome was discovered on the skie but
by the time the whole slide was checked (which only took about 50 seconds), the parasite had died
This systamatic dying of parasites was attributed to the heat of the microscope lamp traumatizing
the parasites, both by the high temperature and the shock of the temperature change. If another
attempt at cloning were to be made, it would be suggested that a11 steps, especially microscopic
examinations, be done at the same temperature, preferably in a cold room.
22
Field Work
Dairy Farms, February 2 1
The Niayes r$gi;on is important for the agricultural and economic development of Senegal
The land has particularly favorable qualities for farming, and the closeness and easy transport of
goods to the export tenter of Dakar makes the development of this region strategic. Within the
Niayes there are two main types of dairy farms. The first are run by the traditional farmers who daily
take their cattle, goats, and sheep to graze on the open land. For generations these local animals
have had considerable exposure to the habitats of tsetse flies (and other biting flies) and ofien have at
least some, if not complete, resistance to trypanosomiasis. The second type of farm is newer and
usually a huge multi-million dollar organization that imports high-milk producing animals such as
Holsteins. Although these cattle show great promise for improving the milk and meat production of
the nation, the animals are usually naive to trypanosomiasis and particularly susceptible during t he
rainy season. Many of these mega farms keep their animals contained and have extensive insecticide
programs to reduce vector-born diseases. Nevertheless, the close proximity of the locally run fàrms
produces a frustrating situation for the mega-farm owners because the untreated, locally grazing
cattle bring home an assortment of diseases afier tramping through the valley. Regardless of the
strict containment of their animals, the large farms are directly affected by the health of the nearby
traditional dairy farts.
In mid-February an FAO officia1 who specializes in trypanosomiasis (Mr. Shizuka) came ao
assess the level of trypanosomiasis risk to dairy animals. Farms in the Niayes region were ofgreatest
concern, following the observation of the tsetse population in an animal park in Dakar.
Niacoulrab. The first farm which was in Niacoulrab, located just before Lac Rose, had 400
cattle (they had just recently reduced their population from :700.) The farm had a rather extensive
breeding program of local breeds with the larger and more milk-producing Holsteins (Annex 13 5).
23
The farm was well-equipped with milk machines, cold rooms, and spray stations. Most of their
cattle produce 30 L per day, with some as many as 36 L during the high season. Although the farm
does not have a serious problem with trypanosomiasis, they still administer prophylactic anti-
trypanosomal drugs to all the animals every 6 months. The greatest risk is during the rainy season,
*,k ’ .1.
.’
3uly-September, when food sources are low and the habitat is more condusive to vector-borne
diseases. Niacoulrab had recently bought some N’Dama cattle, and the veterinarian mentioned some
concern about them carrying trypanosomiasis infections (Annex B.6). This situation was indicative
of the fact that increased abilities to transport animals also enable the spread of otherwise isolated
diseases.
Wayembam. In the second farm we visited, Wayemham, a11 the cattle were Jerseys and had
been purchased from Denmark. They have 120 cattle, with the average production of 30 L per day.
The farm was much less-advanced than Niacoulrab and the cattle are located directly next to a major
route for the local grazing herds (Annex B.7). The veterinarian exprcssed concern for the
transmission of diseases from local animals, and mentioned that he had treated three animais for
trypanosomiasis. He also said that they were planning on building a new, completely enclosed
building for the peak-producing CO~S, as they were always more fragile. The farm’s milk is sold by
women in local kiosks at 500-600CFA, oRen as saur milk as it is not refrigerated (nor homogonized
or treated in any other fashion).
The last large dairy farm we visited was
SOCA.
SOCA, which is located in Sebikotane, very
near the herds in which the Bambilor T.vivax strain was found in September, 1997. SOCA is a
multi-million dollar organization that seemed to be in another class when compared to the two
previous farts. The veterinarian was very well-educated and had a clear concise presentation caf the
farms activities. He mentioned that their greatest problems with disease fa11 during/afIer the rainy
season when the food sources are limited. The soi1 in the area is very fertile, but water sources for
irrigation are difficult. The dairy cows are almost all Jerseys, with a couple other breeds--local and
24
imported. In order to boost their profits, they sel1 milk (raw and pasturized), cream, and juice
(bissap). Their two main customers are big distribution factories as well as the local kiosk and
boutiques. Nearby SOCA there was a strawberry field adjacent to a small area very conducive for a
tsetse fly gallery (Annex B.8).
ARer the dairy farms two other sites were visited as well, neither of which had animals but
were nonetheless interesting for the development of agriculture in the Niaye region. The first being
ENDA Syspro and the second a monastary, the Seminary of Libermann. The seminary had large
fruit tree orchards and had obviously been affected by recent years of drought. Regardless of the dry
climate, some of the monks were emphatic about the presence of tsetse flies on the seminary
grounds.
Park Bandia, March 30
In the beginning of February, 1998, an antelope in the Wild Animal Park Bandia (Annex
B.9,10) died due to a trypanosomiasis infection. Another animal was found dead in the Park in rhe
middle of March, but the cause of death could not be identified because of decomposition. In an
effort to assess the tsetse fly density within the par-k, nine fly traps were set on March 28th.
Two
days later we returned to retrieve the traps and their contents.
There were no tsetse flies or tabanides in any of the traps when we arrived. Of the 9 traps
set, 6 of them were placed along or very near the Somone creek and a11 of these had stomoxyines,
totalling 34. Although there was no bait put in the traps, the high Count of stomoxyines ( 13 in one of
the cages) indicated that the traps were successful and that the tsetse fly population is negligable, if
at ah. As mentioned before, Stomoxyitre are suspected mechanical vectors for trypanosomiasis, and
in an enclosed animal park where animais are more likely to be in close proximity to each other, the
success of mechanical transfer woufd seem to be more probable.
2 5
Sokone, April22-24
As this internship was completed during Senegal’s dry season, the Dakar region and Niayes
valley had very few, if any, tsetse flies. Thus, in an effort to observe a more suitable environment for
trypanosomiasis vectors, a trip to Sokone was planned. On April 22nd we left Dakar with 9 fly
a*:’ *
traps, three mice (Group C: Ml,MZ!,M3), and other supplies,
After arriving in Sokone we drove to the outskirts of the region, where two tsetse fly
galleries were located. The first site was Keur Aliou Gaye (KAG), a dry but classified forest located
adjacent to farming fields. It was divided into two sections, Ai in which we placed two traps (KAG
no 1, no 2) and about 3 kilometers down the road, Aii, where we placed two more (KAG no 3,4).
Both of these stops were very dry and had hard, dry soil. There was quite a bit of vegetation,
although not ver-y green. Aii had a huge monkey residency as well as domestic livestock
Alter driving through many villages we came to the second site, Foret Classe de Patako,
which was a much more moist and humid forest. The fohage was very thick and there were op~n
bodies of water used for irrigating the small plots of vegetables and trees. We placed five traps here
(FCP nol-no5), with much more hope of catching flies. At FCP 2 a fly landed on, and bit, Dr
Diaite’s hand and another one was seen on the back of his shirt (Annex B. 11). On our way out of
FCP, a tsetse fly entered the car and SO we captured and contained it until we arrived back in
Sokone, where it was discovered to be still alive, and a male Glossi~m morsitcms s~~bmorsittrrrs
The next morning we travelled down to the weekly market in Touba Nding, where there
would possibly be livestock infected with trypanosomiasis. We brought the three mice (GroL;p C) in
case a sick animal was found and the owner allowed us to draw blood. As we did not have access to
a microscope, all injections would be blind. Nevertheless, we took blood samples from three horses
and infected the mice. Group C Ml: 0.5 ml injection fiom adult male horse, slightly anemic with
swollen prescapular lymph nodes (Annex B. 12); owner--Modou Mbang, village--Passy Ndenderley.
Group C M2 1 .O ml injection from an adult male horse, slightly anemic with swollen lymph nodes
26
and hind legs; owner--Soulemane Dieng, village--Pakala. Group C M3 1 .O ml injected from a
Young female horse, slightly anemic but not very sick; owner--Moussa Sarr. In the afiernoon we left
for the tsetse fly sites which produced the following results:
Keur Aliou Gave
.
Foret Classe de Patako
,>‘ q.
IL4G 1: 9 Stomoxyines, appx 10 other flies
FCPI: lfly
KAG 2: 1 bee
FCP 2: 2 Glossine (1 m + 1 f), 2 Stomoxyines
KAG3: 1 wasp
FCP 3: 10 Glossine (5m + 5f)
KAG 4: 2 Stomoxyines, appx 4 other flies
FCP 4: appx 10 flies
FCP 5: 4 Glossine (3m -t lf), 5 Stomoxyines
When we arrived in Sokone, ah the flies were caref?rlly observed and rechecked. Al1 the tsetse fies
were identified as G.morsitans submorsitans as indicated by their “black sock” legs. Because KAG
was SO dry and unlikely to have tsetse flies, it was disregarded when calculating fly density. Thus
given the five traps at FCP, the overall density was calculated to be 3.2 flies per day, per trap
‘l‘his
fly density is relatively high, especially considering that it was during the dry season. Although
Glossinapalpalis were not found in any of the traps, it would be expected that they would be
present during the rainy season. G.palpalis need a more moist, humid climate and tend to follow
very closely to water ways. When the weather is drier, G.palpa1i.s Will retreat to pockets of humidity
and the G.morsitans Will retreat to the areas previously inhabitated by the G.palpalis.
If the chmate
is wet enough, G.morsitans Will abandon major water ways and spread out into the region. Such
migration habits affect not only the fly densities, but more importantly the two species’ host
selection.
27
fumex
28
P
0 ih
w
Kl in
1
A.
N
29
Annex
Parasiternia Level
--L in
--L
0
in
0
P b
P
cd 67
w
!Q m
Annex A.2
Parasiternia Level
rd
f
2 VI
2
0
u1
0
2y
5
8
11
14
17
20
23
26
29
32
35
38
41
44
47
68
71
74
77
80
83
86
89
92
95
98
101
104
107
n
Annex B. 1
Annex ES.2
31
Annex B.3
Annex B.4
32
Annex B.5
Annex B.6
33
. . . . .--
Annex B.7
Annex B.8
34
Annex Es.9
Annex B.10
35
Annex B. 1 Ii
Annex B.12
36
Bibliog-aphy
1) Balaban, N., Waithaka, H.K., Njogu, A.R., Goldman, R. Intraceliular Antigens (Microbutule-
Associated Protein Copurified with Glycosomal Enzymes)-Possible Vaccines against
Trypanosomiasis. The Journal of Infections Diseuses 172 (1995) 845-850.
y; ”
2) Baltz, T., Baltz, D., Giroud, C., Crockett, J., Cultivation in a semi-defined medium of animal
infective forms of Trypanosoma brzrcei, T. eqztiperdzrm, T evansi, 11 rhodesiense, and
Tgambiertse. ne lZkB0 Journal. 4 (1985) 1273-1277.
3) Diaite, A., et al. Work submitted Februarly 1998
4) Ekwanzala, M., Pepin, J., Khonde, N., Molisho, S., et al. In the heart ofdarkness: sleeping
sickness in Zaire. The Latzcet 348 (1996) 1427-l 430
5) Gouteux, J.P., Kounda Gboumbi, J.C., Noutoua, L., et al. Man-fly contact in the Gambian
trypanosomiasis focus of Nola-Biolo (Central African Republic). E-opical Medicine at~d
Parasitology. 44 (1993) 213-218
6) Hirumi, H. And Hirumi, K. Continuous Cultivation of ‘Ijl~~atzosoma brzrcei Blood Stream Forms
in a Medium Containing a Low Concentration of Serum Protein Without Feeder Cell Layers
ïhe Journal of Parasitology. 75 (1989) 985-990.
7) Hoare, Ceci1 A. The Trypanosomiasis of A4mr~rncr1.s
1972 Blackwell Scientific Publications
8) Kuzoe, F.A.S., Current situation of African trypanosomiasis Acta Copica 54 (1993) 153- 162
9) Leak, S.G.A., Peregrine, A.S., Mulatu, W., Rowlands, G.J., and D’leteren, G. Use of insecticied-
impregnated targets for the control of tsetse flies (G/o.wirm spp.) and trypanosomiasis
occurring in cattle in an area of south-west Ethiopia with a high prevalence of drug-resistant
trypanosomes. 5 (1996) 599-609.
10) Lumsden, Z.H.R. Trypanosomiasis. Zmmunokogy. 2 ( 1978) 1492- 1499.
11) McGovern, T.W., Williams, W., Fitzpatrick, J.E., Cetron, M.S., et al. Cutaneous manifestations
of Afiican trypanosomiasis. Arichives of Dermatology. 13 1 ( 1995) 117% 1182
12) Murray, M., Trail, J.C.M., Turner, D.A., and Wissocq, Y. l’roductivite Animale et
Tkypanotolerance.
1983, 9.
13) Winrock International. Assesment of Animal Agriculture itl ,Crrb-Saharan Africa. 1992
14) http://www.urmc.rochester.edu/smd/mbi/cann/224/Trypano.html
15) Jennings, F.W., Atougia, J.M., Murray, M. Topical chemotherapy for experimental murine
Alï-ican CNS-trypanosomiasis: the successful use of the arsenical, melarsoprol, combined
with the 5-nitroimidazoles, fexinidazole or MK-436. Tropical Medicine and lnternatiorral
HeaIth. 5 (1996) 590-598
3 7