African Journal of Biotechnology
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African
Journal of Biotechnology Vol. 1 (2), pp. 39-45, December 2002 ISSN 1684-5315 © 2002 Academic Journals
1Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Roslin, Midlothian, Scotland, EH25 9RG, UK 2Livestock Health Research Institute, PO Box 96, Tororo, Uganda 3University of Glasgow Veterinary School, Bearsden Road, Glasgow, Scotland
Accepted 12 November 2002 |
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| Abstract | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Reliable
DNA based methodologies to determine prevalence of trypanosome species in
domestic livestock have been available for over 10 years.
Despite this, they are rarely used to generate baseline data for
control operations for these diseases in the field.
Rather, such operations tend to rely on data which can be generated
using low technology methods such as direct observation of parasites by
light microscopy. Here we
show the pitfalls of relying on such low tech methodology which, although
simple in its application, can provide inaccurate and inadequate data on
which to base control methodologies. Our
analysis of 61 cattle selected for trypanosome carrier status by either
microscopy, low PCV or poor condition score, showed that 90% were
infected with trypanosomes while 84% of the total were infected with T. brucei. Diagnosis by PCR on buffy coat preparations on Whatman®
FTA®
matrices was the most sensitive methodology relative to the gold standard,
whereas microscopy was the least sensitive.
Keywords: sleeping sickness, human African trypanosomiasis, epidemiology, chemotherapy, PCR.
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| Introduction | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Human
African trypanosomiasis (HAT) or sleeping sickness, a disease thought to
have been largely conquered during the 1960’s, has re-emerged as a
serious public health problem over large swathes of sub-Saharan Africa (Welburn
et al., 2001).
It has been estimated that 300,000–500,000 people are currently
infected and 100,000 deaths are caused each year by the disease (Cattand et
al., 2001).
HAT is strictly a problem for rural communities in Africa since it
is dependent for transmission on tsetse.
The disease persists in areas which have suffered most acutely from
general breakdown of infrastructure, including health care (Louis, 2001).
The
cause of human sleeping sickness in eastern Africa is Trypanosoma
brucei rhodesiense, a zoonotic parasite, which is mainly a
non-pathogenic parasite of livestock and wild bovids (Heisch
et al., 1958; Onyango et
al., 1966)
but which
results in sleeping sickness when transmitted to humans.
Control of T. b. rhodesiense
HAT is complicated by the fact that T.b.
rhodesiense co-exists in domestic livestock with the morphologically
identical Trypanosoma brucei brucei
which is not pathogenic to humans. Furthermore,
in the regions where HAT prevails, several other trypanosome species,
including T. vivax and T. congolense, are prevalent which affect the health of cattle and
other livestock. Taken as a
group, the trypanosomiases of livestock are responsible for severe losses
in the agricultural sector (Kristjanson, 1999),
and together with the human disease burden imposed by human sleeping
sickness (Odiit et al.,
personal communications), the trypanosomiases more generally form a very
significant group of parasitic infections. Quantifying
the risk presented to rural peoples by T.b.
rhodesiense infected
livestock and wild animals is essential in determining the extent to which
control activities are required and the control methodology which is
appropriate. Of particular importance to public health is this new
possibility of rapidly identifying animals that are serving as reservoirs
for human infective trypanosomes. The
risk of spreading sleeping sickness is closely related to the movement of
cattle populations, and the screening and treatment of livestock to
prevent disease spread has been advocated (Fèvre et al.,
2001). This was until recently impossible, as there
was no means of safely differentiating this human infective parasite from
morphologically identical T. b. brucei. Recently, however, it was observed that a strain of T. b.
rhodesiense belonging to the Busoga zymodeme (noted for its
persistence over time in southeast Uganda) had a gene (serum resistance
associated gene, SRA), co-expressed with the variant antigen gene,
which was shown experimentally to confer human serum resistance (Xong et al., 1998).
This gene has been shown to unequivocally differentiate T. b.
brucei from T. b. rhodesiense in southeast Uganda.
SRA has been found in all human isolates of T. b.
rhodesiense identified in southeast Uganda to date and moreover can
confirm the human infectivity status of trypanosomes isolated from
animals. SRA is not present in those samples previously designated
as T. b. brucei by restriction fragment length polymorphism
analysis (Welburn et al.,
2001b; Welburn
and Odiit, 2002).
Although
these molecular diagnostic tools are proving extremely useful for
elucidation of sleeping sickness and animal trypanosomiasis epidemiology,
they are also of practical use in terms of better targeting drug use in
animals to improve animal health.
Perhaps most importantly, the molecular tools allow the identification of
individual reservoir hosts carrying human-infective T. b. rhodesiense parasites.
Despite this, however, more traditional technologies are still in
widespread use, particularly microscopy-based diagnosis (reviewed
in Uilenberg, 1998).
In this paper, we compare different methodologies used to prepare parasite material in the field for the eventual assessment of trypanosome-infection status of the animals, and thus for the risk posed by those animals to disease in cattle-keeping populations. The more accurate the diagnosis of infections can be, the more targeted interventions can become and the greater the reduction in the risk of human disease. Selective treatment of infected animals in this way results in a holistic approach to disease control, concurrently combating livestock and human disease agents, and resulting in an overall improvement in the livestock and human health of rural populations.
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| Materials and Methods | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Sampling In
2001, as part of a large survey of trypanosome infections in the cattle
population in the sleeping sickness focus in Soroti district, eastern
Uganda (Fevre et al., 2001), 150 animals were
subjected to clinical examination and screened for trypanosomes by two
standard field methods, the Buffy Coat technique, BCT (Murray et. al., 1977)
and using the haematocrit centrifugation technique, HCT (Woo, 1970). Animals
were selected for the study according to either a
positive identification
of carrier
status by microscopy,
a low PCV or a poor observed condition score (Nicholson & Butterworth,
1986) following Duvallet
et al. (1999)
who showed that 50% of parasitologically negative animals with a
haematocrit value under 25% were subsequently identified as trypanosome
positive using PCR.
Ten millilitres of blood was withdrawn into a heparinised
vaccutainer from the jugular vein [Solano et al. (2002) showed heparin was less
inhibitory to PCR reactions than EDTA]. Sample
preparation for PCR in the field Three
methods of sample preparation were assessed for sensitivity analysis for
PCR determination of T. brucei: direct DNA preparation using DNAzol; application of whole
blood to Whatman FTA cards; application of buffy coat preparations to FTA
cards. Direct
DNA preparation DNA
was directly isolated from 1ml of bovine blood using DNAzol® BD
Reagent (Chomczynski et
al., 1997).
DNAzol “is a complete, non-toxic and ready to use reagent for the
isolation of genomic DNA from various biological sources”.
Whole blood was mixed at a ratio of 1:2 with DNAzol® BD
prior to the addition of isopropanol.
The sample was shaken vigorously and left at room temperature for 5
minutes. Precipitated DNA was
isolated by centrifugation at 6000 g for 6 minutes and stored at -20°C
prior to further processing. Whole
blood application to FTA® Cards One
hundred microlitres of whole blood was applied directly onto FTA®
Cards (Whatman®) which were allowed to dry thoroughly prior to
storage at room temperature. Buffy
coat preparations onto FTAÒ
Cards One
ml whole blood was centrifuged at 10,000 g for 5 minutes.
100ml
of buffy coat was withdrawn and applied directly on the FTA® Cards. Preparation
of DNA for PCR analysis DNAzol® BD lysate:
The DNA pellet was washed with 0.5ml of reagent buffer, mixed to ensure
complete dispersal, and centrifuged at 2000g for 4 minutes. The supernatant was removed and the pellet washed with 1ml of
95% ethanol. DNA was recovered by centrifugation and solubilised in 66ml
of 8mM NaOH, the solution neutralised after 5 minutes with the addition of
33ml
of HEPES. FTA® Card:
Two mm
discs were cut from the sample-saturated cards and prepared according to
the manufacturers instructions. Briefly,
the discs were washed twice in FTA purification reagent to remove any PCR
inhibitors from the sample, and traces of FTA buffer were removed by two
further washes in TE buffer. The
discs were air dried, then transferred to PCR tubes where they were used
to seed the amplification reaction. Trypanosome species specific
PCR DNA
prepared using DNAzol, and bound to FTA matrix (from blood of buffy coat
source) were assessed for the presence of the trypanosomes which may be
present in domestic cattle; namely Trypanozoon, Duttonella and Nannomonas
(four types of Trypanosoma congolense). The
primers used were supplied by Sigma (see Table 1).
Table 1. Primer sequences and amplification product size.
PCR
reaction conditions and amplification protocols Standard
PCR amplifications were carried out in 25ml
reactions mixtures containing the final concentrations, 10 mM TrisHCL pH
8.3, 50 mM KCl, 1.5 mM MgCl2, 200 mm
of each of the 4 deoxynucleoside triphosphates and 1 Unit of REDTaq DNA
polymerase (Sigma). Genomic
template preparations of 1 ml
DNAzol purified DNA or 1x2mm FTA card discs were amplified.
Specific PCR conditions in terms of primer concentrations and
amplification programmes are listed on Table 2, a final elongation step of
5 minutes was incorporated into each programme. PCR products were
separated by electrophoreses in a 1.5% (w/v) agarose gel containing 0.5 mg/ml
ethidium bromide and visualised by ultraviolet light.
Table 2. Species specific PCR conditions and programmes.
Defining any trypanosome species infection It was assumed that each test was 100% specific (i.e. identification by microscopy or detection or amplification of DNA primers was indicative of trypanosome presence). Therefore, the gold standard, or true infection status of each of the 61 cows, was defined as positive if at least one of the four diagnostic tests were positive. Defining T.
brucei species infection Because of the difficulty of distinguishing trypanosome species with microscopy, the gold standard, or true T. brucei species infection status for each of the 61 cows, was defined as positive if a least one of the three DNA tests were positive. Statistical analysis For each of the four tests, the sensitivity (and 95% confidence intervals) was calculated relative to the gold standard status, for both infection with any trypanosome species and infection with T. brucei species. Differences between the sensitivity of the different tests were examined using the c2 test. The kappa statistic (and 95% confidence intervals), which gives a measurement of the degree of agreement between each of the four tests and the gold standard, was also calculated, again for infection with any trypanosome infection and infection with T. brucei species. The value of kappa lies between 0 and 1, with 1 meaning perfect agreement between tests and 0 meaning that the association is no better than expected from chance alone. All statistical analyses were performed in SPSS (release 10.0.5). |
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| Results | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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A total of 61 cows were diagnosed using the four tests. A summary of the results is shown in Table 3. Overall, 55 (90.1%) of the cows had a trypanosome infection with 51 infected with T. brucei species. The sensitivities of the four different tests, for any trypanosome infection and T. brucei species specifically, relative to the gold standards (defined above) are shown in Figure 1. Statistical comparisons of the test sensitivities are shown in Tables 4a (for any trypanosome species detection) and 4b (for T. brucei species detection). The measurements of agreement between each of the four diagnostic tests and the defined gold standard are shown in Figure 2.
Table
3. Summary of positive
diagnostic results.
*includes one cow that was identified as positive for T. brucei species with microscopy but negative on all three PCR test. The cow was shown by PCR to have a T. vivax infection.
Figure 1. Sensitivity of the four diagnostic tests relative to the gold standard.
Figure 2. Measure of agreement (kappa statistic) between each of the four diagnostic tests and the gold standard.
Table 4a. Comparison of the test sensitivities for any trypanosome species-detection using the c2 test
Table 4b. Comparison of the test sensitivities for T. brucei species detection using the c2 test.
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| Discussion | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Molecular
methods for the species-specific identification of trypanosomes have been
available for nearly 10 years (Masake et
al., 1994, 1997; Majiwa et
al. 1993, 1994).
Despite this, their application in disease control and research
programmes in the field has been limited to a handful of studies on cattle
like that of Clausen et al.
(1998) on naturally infected dairy cattle in peri-urban
Kampala, Solano et al. (1999) in Burkina Faso
and Mugittu et al. (2001) in Tanzania.
The more traditional methods involving microscopy have prevailed in
most field studies, despite the fact that it has been suspected for some
time that these classical methods are missing a large proportion of the
infections present in livestock, due to the presence of
trypanosome-positive DNA signals from cross-checked cattle deemed
aparasitaemic by these methods (Majiwa et
al., 1994).
The principal problem with microscopy is one of sensitivity. The
application of DNA techniques provides much improved levels of
sensitivity, such as the possibility of detection of individual organisms
in samples of whole blood. Microscopy
remains useful particularly as it can be carried out directly at the field
level and gives immediate results. The
original molecular protocols were not immediately applicable to conditions
in the field, due to the amount of processing required and the time
involved prior to the testing of the samples themselves.
However, recent developments in methods of extracting and/or
preserving DNA have changed this, making sample collection with a view to
diagnosis by PCR more realistic at the field level.
In
this study, we compared the diagnostic sensitivity of classical
parasitological examination by microscopy against DNA methodologies –
PCR on blood samples treated with DNAzol and PCR on blood and buffy coat
samples collected on FTA cards, to evaluate which methods provides the
greatest sensitivity for trypanosome identification and species diagnosis.
The direct preparation of DNA using DNAzol direct purification (Chomczynski
et al., 1997)
uses guanidine thiocyanate and a detergent mixture. The recent development
of the Whatman® FTA® matrix offers significant
advantages for sample collection for the preservation of DNA (Hsiao et al., 1999).
These FTA matrices have been successfully tested for whole blood
storage and malaria diagnosis by PCR (Zhong et
al., 2001) and Dobbs et
al. (2002)
found that tumour cells stored on an FTA matrix performed as well in PCR
as freshly extracted DNA in over 95% of cases. Samples stored on this
matrix have the added advantage of a 10-year shelf life at room
temperature. Here,
we have shown that PCR following application of buffy coat to the FTA
matrix is the most sensitive diagnostic test for all trypanosome
species-specific PCR reactions currently available.
There was no significant difference in
the sensitivity of PCR
on whole blood applied to the FTA matrix and whole blood DNA extraction
with DNAzol for all trypanosomes, and all these methods are a significant
improvement on the sensitivity offered by the standard HCT and BCT
microscopy methods for all trypanosomes.
However, the results show that PCR on whole blood on the FTA matrix
is not significantly different from microscopy for T.
brucei alone, suggesting that, for the accurate determination of T.
brucei prevalence in Zebu cattle, a concentration step (preparation of
buffy coats) is necessary. Given
that in this region (Soroti, eastern Uganda) 40% of cattle with T.
brucei infections were also positive for the human serum resistance
gene (Welburn et al., 2001b), sensitive and accurate
detection of T. brucei infections in cattle is essential for planning control
programmes for human African trypanosomiasis.
These findings are consistent with earlier studies of Clausen et
al. (1998) who demonstrated that the detection
rate by PCR was two times higher than the detection rate with
parasitological techniques, and that of Solano et
al. (1999)
who showed that PCR on buffy coat samples for diagnosis is more sensitive
than on whole blood extracted from filter papers using Chelex, as the
concentration of trypanosomes is higher in the buffy coat.
Furthermore, Mugittu et al. (2001) found that trypanosomes could be
detected in 43% of parasitologically negative cattle (62 samples) using
PCR. Given
these improved tools, it becomes apparent that there are far more
trypanosome-infected cattle circulating in the herds in tsetse infested
regions than previously thought, and that control programmes which have
been designed around prevalence information gained from microscopy studies
may have under-estimated the degree of the problem.
In terms of animal herd and human health, the aim of such
programmes is the control of parasites in the reservoir of infection.
When the extent of this reservoir is not properly accounted for due
to the poor sensitivity of the tools used, the resulting interventions
will not be adequate. Screening
of animals is a necessary step to assessing, and ultimately reducing, the
risk of outbreaks of the trypanosomiases in previously unaffected areas (Enyaru,
1999; Fèvre et
al., 2002).
Sustainable
control programmes must also consider ways of eliminating or limiting the
spread of the trypanosome reservoir of disease through domestic livestock
populations. One solution would be to treat cattle at point of sale in the
markets. Controlling the reservoir of parasites of all species is
essential (Welburn and Odiit, 2002) while
bearing in mind the longer-term problems that large-scale chemotherapy of
cattle may pose in terms of trypanocidal drug resistance (Barrett, 2001).
ACKNOWLEDGEMENTS This
work was funded by the Cunningham Trust and the Animal Health Programme of
the Department for International Development (DFID) of the United Kingdom.
The views expressed are those of the authors and not necessarily those of
DFID.
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