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Lymbery, A.J . and Thompson, R.C.A. ( 2012) The molecular
epidemiology of parasite infections: Tools and applications.
Molecular and Biochemical Parasitology, 181 ( 2) . pp. 102-116.
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The molecular epidemiology of parasite infections: tools and
AJ Lymberya,b*, RCA Thompsonb
aFish Health Unit and bWHO Collaborating Centre for the Molecular Epidemiology of Parasitic Infections,
School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch WA 6150, Australia
*Corresponding author. Tel +64 8 93602729; Fax +64 8 9360 7512.
E-mail address: email@example.com
Molecular epidemiology, broadly defined, is the application of molecular genetic
techniques to the dynamics of disease in a population. In this review, we briefly describe
molecular and analytical tools available for molecular epidemiological studies and then
provide an overview of how they can be applied to better understand parasitic disease. A
range of new molecular tools have been developed in recent years, allowing for the direct
examination of parasites from clinical or environmental samples, and providing access to
relatively cheap, rapid, high throughput molecular assays. At the same time, new
analytical approaches, in particular those derived from coalescent theory, have been
developed to provide more robust estimates of evolutionary processes and demographic
parameters from multilocus, genotypic data. To date, the primary application of
molecular epidemiology has been to provide specific and sensitive identification of
parasites and to resolve taxonomic issues, particularly at the species level and below.
Population genetic studies have also been used to determine the extent of genetic
diversity among populations of parasites and the degree to which this diversity is
associated with different host cycles or epidemiologically important phenotypes. Many of
these studies have also shed new light on transmission cycles of parasites, particularly the
extent to which zoonotic transmission occurs, and on the prevalence and importance of
mixed infections with different parasite species or intraspecific variants (polyparasitism).
A major challenge, and one which is now being addressed by an increasing number of
studies, is to find and utilise genetic markers for complex traits of epidemiological
significance, such as drug resistance, zoonotic potential and virulence.
Keywords: molecular epidemiology; parasite identification; species delimitation;
transmission; genetic markers
1. What is molecular epidemiology?
Epidemiology is the study of the causation and dynamics of disease in a population. For
parasitic diseases, this is determined by the transmission of the parasite between hosts,
and how this transmission affects the dispersal of the parasite within and among host
populations . To control parasitic disease, therefore, we need to understand parasite
ecology, particularly transmission dynamics, how life cycles may interact and the nature
of interactions within the host. This requires an input from both population and
evolutionary biology, to determine, for example, the genetic structure and evolution of
infectious agents, their population biology, and the evolutionary consequences of medical
and public health interventions .
Traditionally, both epidemiology and parasite ecology have concentrated on an empirical
approach. Epidemiological studies typically begin with a description of the frequency and
distribution of disease and then attempt to associate these patterns with the frequency and
distribution of independent variables or risk factors. Identifying risk factors is important
because it allows for targeted control programs, but the efficacy of such control programs
hinges upon knowing how the risk factors interact with the parasite’s life cycle to
increase exposure. Ecological studies of parasite life cycles usually start with a
description of parasite prevalence, and sometimes also intensity of infection, within
different host species. These data, often accompanied by in vitro and experimental
infection studies, can then be used to infer the major pathways of parasite transmission.
In the last 20 years, these empirical studies of the ecology of parasite life cycles and the
epidemiology of parasitic disease have been complemented by a more theoretical
approach, which uses mathematical models of parasite and host population sizes to guide
epidemiological interpretation [e.g.3,4,5].
The application of molecular and analytical tools, derived largely from the fields of
population genetics and systematics, can contribute enormously to both empirical and
theoretical studies of the epidemiology of parasitic disease. Molecular epidemiological
approaches enable the reconstruction of evolutionary relationships between parasites over
a wide range of temporal and spatial scales, improving our ability to identify parasites,
track their movements, relate their spread to environmental factors and understand the
role they play in disease causation .
In this review, we briefly describe the range of molecular and analytical tools available
for molecular epidemiological studies and then provide an overview of how they can be
applied to better understand the causation and dynamics of parasitic diseases. Tibayrenc
[7,8] suggested that molecular epidemiological studies of parasitic diseases could be
classified into two different types, depending on whether they were concerned purely
with identification of the causative agents of disease, or whether they considered the
impact of genetic variation on “downstream functions”, such as transmission, infectivity,
virulence or drug resistance. To date, the main applications of molecular techniques have
been in parasite identification rather than to study patterns of disease progression or
transmission. This has been due partly to the inability of molecular tools to distinguish
genetic variation at the appropriate level of resolution for addressing downstream
function and partly to the inadequacy of analytical methods to interpret genetic variation
in an ecologically meaningful fashion. Rapid advances in both these areas mean that that
an increasing number of molecular epidemiological studies are addressing questions of
function, although parasite identification remains a critical issue. From a practical
perspective, genotyping the agents should not become a dominant aim of molecular
epidemiological investigations, since the existence of different genotypes does not imply
they necessarily have some phenotypic importance. The scope and potential of molecular
epidemiology is much greater, and in this regard the search for genetic markers for
‘medically’ important traits such as infectivity, drug resistance and virulence present
important challenges for molecular epidemiological investigations.
2. Molecular tools
Our ability, using molecular techniques, to detect and characterise the genetic variability
of infectious agents, particularly at the intraspecific level, can be seen as the foundation
for most molecular epidemiological studies . The application of appropriate molecular
tools will aid in the identification and surveillance of infectious agents and in determining
sources of infection. The availability of such tools, particularly those based on the
polymerase chain reaction (PCR), which allow direct examination of clinical or
environmental isolates, has had an enormous impact on the genetic characterisation,
diagnosis and taxonomy of parasites. They also obviate the need for laboratory
amplification of parasite isolates, which was a major limiting factor in characterising
parasites refractory to in vitro culture, and may lead to biased sampling of natural
diversity by the selective amplification of those parasites amenable to culture.
Using PCR, defined gene sequences of infectious agents can be detected from small
quantities of material and the resultant data can be used not only for diagnosis, but also to
assess the effect of interventions on the population structure of infectious agents,
assessment of intraspecies diversity, and transmission studies. The value of such tools is
greatest if they can be applied directly to faecal or tissue specimens, as well as
environmental samples, and if there is the potential to automate such procedures. Table 1
summarises the available molecular tools and their application. Emphasis will be given in
the future to establishing high throughput molecular assays such as pyrosequencing, as
well as their field applicability. Pyrosequencing techniques have the added advantage of
allowing the simultaneous detection of multiple species/genotypes in a single sample
[10,11,12]. Multiplex PCR (mPCR) also enables the amplification of more than one
target of interest in a PCR by using multiple primer pairs and producing amplicons of
different size . Loop-mediated isothermal DNA amplification (LAMP) is a newly
developed, rapid, quantitative, highly sensitive and specific nucleic acid-based, non-PCR
diagnostic tool [14,15,16], applicable to ‘low-cost’ laboratory settings. This simple
molecular test can be carried out on a bench with a heating block instead of a thermal
cycler and may prove to be an invaluable ‘field friendly’ tool for screening and
quantifying infections in host populations while providing important genotypic
Choosing an appropriate marker for molecular epidemiological studies requires
consideration of the required level of resolution for the study, the precision of the genetic
data collected and the historical information content of the data.
Genetic markers, although identified in individuals, are influenced by processes which
are more readily measured at the level of populations, such as mode of reproduction,
breeding system, mutation, migration and selection. Population level processes eventually
influence speciation and thus all cladogenetic events in the history of a lineage.
Therefore, by using genetic markers with appropriate rates of change, we should be able
to examine evolutionary patterns and processes at all levels throughout the hierarchy of
life, from individuals to kingdoms. In this context, emphasis has been given to the
importance of appropriate analysis and the value of characterising the genetic diversity of
infectious agents at different levels of specificity [18,19,20]. The latter requires choosing
molecular tools which are capable of discriminating genetic variants at different
hierarchical levels and the region of DNA examined must be appropriate to the level of
questions being asked [13,20,21,22,23]; e.g. taxonomy, diagnosis, population genetics,
evolutionary relationships, isolate tracking etc. (Table 1). This is primarily a question of
choosing a genomic region with an appropriate signal to noise ratio; too little variation
will provide a signal which is too weak to discriminate among groups, whereas too much
variation will swamp the signal with uninformative noise.
The choice of genetic marker typically involves a trade off between technical
convenience and precision. Markers such as RAPDs and AFLPs do not require specific
sequence information from the target genome, and hence can be utilized more readily for
less well studied species. The variation they detect, however, may be non-heritable and
even when heritable is dominant rather than co-dominant. This means that alternative
alleles at a locus cannot be distinguished, greatly reducing the range and power of
analytical techniques which can be applied to the resultant data. It also means that the
data cannot be compared effectively over different studies, and are therefore limited in
usefulness to the particular time and place where they were collected.
Traditional genetic markers, such as allozymes and RFLPs have little historical
information content. That is, we do not usually know the phylogenetic relationship
between alternative alleles or haplotypes and the data are therefore analysed as allele or
haplotype frequency differences among groups. Sequence data, however, do provide
historical information because the phylogeny of sequences can usually be inferred. This
enables sequence data to be analysed in ways which are not possible for allele frequency
3. Analytical tools
Concomitant with the development of new genetic markers and the ability to rapidly
genotype large numbers of genetic loci has been the development of new analytical tools
to interpret these multilocus genotypes, and a blurring of the boundaries between
population genetic and systematic analyses. Traditionally, these fields have been quite
distinct in their analytical approaches. Population genetics aims to describe and
understand the processes underlying the distribution of genetic variation within and
among populations of the same species, while systematics aims to describe and organize
the pattern of evolutionary relationships among species and higher taxa. For sexually
reproducing organisms, evolutionary relationships above and below the species level are
quite different in nature . Below the species level, relationships between genes
sampled from different individuals are not hierarchical because homologous genes from
the two parents combine in their offspring. Above the species level, however,
relationships between genes sampled from different taxa are hierarchical because they are
a consequence of speciation followed by long periods of reproductive isolation. Methods
developed for inferring phylogenetic relationships above the species level rely on
assumptions that are often violated by the reticulate relationships between individuals
below the species level. Population genetic analyses, therefore, have not traditionally
considered genealogical relationships among genes or among individuals, and systematic
analyses have traditionally ignored the possibility of reticulate relationships. This,
however, is now changing, driven in part by the availability of DNA sequences and other
molecular markers with historical information content and in part by increased computing
power, which makes more feasible the application of statistical techniques such as
maximum likelihood and Bayesian methods .
3.1. Systematic analyses
In recent years, there has been an increasing interest in representing phylogenetic
relationships above the species level as networks rather than as strictly bifurcating trees
[26,27]. This is partly because of the desire to present character conflict or uncertainty in
the reconstructed phylogeny, even when the true evolutionary relationship is believed to
be hierarchical, and partly because of the recognition that evolutionary events such as
hybridisation, horizontal gene transfer and symbiosis may create true non-hierarchical
relationships. Character conflict is often represented by a split network, where parallel
edges connect the nodes, while reticulate networks, where some nodes have more than
two parents, are often used as an explicit representation of complex evolutionary events
. A number of different network construction methods are available, including
median networks , neighbour-nets  and reticulograms .
3.2. Population genetic analyses
The traditional approach to population genetic analysis is based on allele frequencies,
without regard to historical relationships between these alleles. To infer the action of
evolutionary processes, such genetic drift, migration or selection, allele frequencies are
compared with equilibrium expectations, derived from particular models of population
structure. More recent studies have incorporated explicit tests of the effects of landscape
heterogeneity on evolutionary processes, an approach known as landscape genetics
[31,32]. Although this approach has proved very powerful and yielded important insights
into the ecology of parasites and the epidemiology of parasitic diseases, it also has
important limitations. First, when applied to genetic markers such as DNA sequences and
microsatellites, it does not utilize their historical information content. Even worse, if the
markers are extremely variable, allele frequencies become meaningless because every
sequence is different. Second, the equilibrium assumptions upon which many analyses
depend are not always valid. This is a particular problem for parasites of people and
domestic animals which may be subjected to rapid, long distance dispersal as a
consequence of host movements. Third, traditional population genetic analyses require an
a priori demarcation of breeding populations or demes. This is not always
straightforward, especially for parasites which must leave the host to complete their life
Assignment methods, which use information from genetic markers to ascertain
population membership of individuals or groups of individuals, have the potential to
overcome some of the limitations of traditional allele frequency approaches . In
particular, assignment methods do not require that a stable equilibrium has been achieved
between opposing evolutionary forces (although they do usually assume that the
population is in Hardy-Weinberg and linkage equilibrium), so they are often more
appropriate for parasites with recent history of invasions or range expansions. Despite
their advantages, there are limitations to the use of many assignment methods with
parasite species. In particular, assumptions of Hardy-Weinberg and linkage equilibria
limit their usefulness for species which do not reproduce sexually or have high rates of
inbreeding. They also may not distinguish population subdivision from other processes
such as small population size, inbreeding and genetic bottlenecks, which may cause
departures from Hardy-Weinberg and linkage equilibria.
Phylogenetic methods make use of intraspecific gene genealogies rather than allele
frequencies to infer evolutionary processes, usually in a geographical context
(phylogeography). The great strength of phylogenetic methods of analysis is that they add
a temporal dimension which can be related to spatial organization among alleles .
Important information can be obtained from intraspecific gene phylogenies even in the
absence of a population genetic model [e.g. 34], but an explicit population genetic model
provides extra power to test specific hypotheses about evolutionary processes .
The two main approaches to inferring evolutionary processes from a reconstructed
intraspecific phylogeny are nested clade analysis and coalescent-based methods,
particularly approximate Bayesian computation. Nested clade analysis is a method of
inferring the role of contemporary evolutionary processes, such as gene flow, and
historical events, such as population fragmentation, range expansion or colonization,
from the geographic structure of intraspecific gene clades [36,37]. Briefly, a series of
hierarchically nested clades are defined from the phylogeny. The geographic distributions
between clades at different hierarchical levels are statistically compared and the pattern
of the comparison is used to test hypotheses about evolutionary processes which have
been developed from simulation models. Nested clade analysis, while it has been applied
to infer evolutionary processes responsible for genetic structure in parasites [e.g. 38], has
been widely criticized for the subjective nature of the inference process [39,40; although
Coalescent approaches to population genetic analysis are based on coalescent theory,
which is a mathematical description of the genealogical history of a sample of neutral
alleles from a population. A pivotal result from coalescent theory is that coalescence
time, the time at which two alleles share a most recent common ancestor, is a function of
the demographic history of the population . In theory, this enables a likelihood
function to be calculated, which considers both the probability of obtaining the observed
data given an intraspecific phylogeny, and the probability of the phylogeny given certain
genetic or demographic parameters . Calculating the likelihood is not usually
computationally feasible and instead it is approximated by a variety of techniques. The
most widely used technique is approximate Bayesian computation, in which the data are
compressed into summary statistics and calculation of the likelihood is replaced by a
comparison of observed and simulated data . These methods have not been utilized
widely for parasites, probably because models relating gene phylogenies to evolutionary
processes are complicated by the need to consider the epidemiology of parasitic infection.
For microparasites, where successful reproduction requires both replication within hosts
and transmission between hosts, gene phylogenies in a component population (all the
members of a parasite species in all the individuals of a particular host species) composed
of many infrapopulations (all the members of a parasite species within a single host
individual), should be reasonably approximated by standard metapopulation models .
Different patterns of immune response in the host, however, can produce quite different
gene phylogenies in the parasite  and will have to be explicitly incorporated in
coalescent models to infer genetic parameters. For macroparasites, the metapopulation
model is complicated by the extent to which parasite infrapopulations have a stable
recurrence of generations, which is determined by the extent of correlated transmission of
offspring from one host to the next (; see also section 4.1.2).
4. Identification and classification of the causative agents
Parasite control depends upon the rapid, accurate detection and identification of the
aetiological agents, so that cycles of transmission can be inferred and the potential for
interaction between cycles determined. Effective control also requires the ability to
characterise parasites from different stages in their life cycles in tissues, blood, faeces or
the environment, on the basis of epidemiologically useful features. These include host
specificity, public health significance in terms of zoonotic potential, virulence and drug
sensitivity. Traditional diagnostic techniques involving microscopy have thus been
complemented by a variety of molecular tools that provide additional information about
the causative agents.
Molecular identification is particularly important when discriminating different parasites
with morphologically identical life cycle stages, such as eggs or cysts, from faecal
samples, or when attempting to match different life cycle stages of the same parasite from
intermediate and definitive hosts . For example, it is now emerging that in some
endemic areas humans may be infected with more than one species of hookworm, and the
eggs expelled in the faeces are morphologically identical . Fortunately, PCR-based
procedures have been developed which can differentiate between all the relevant genera
and species of hookworm of public health and veterinary significance . It is
important to be able to distinguish between the two main genera of human hookworm,
Ancylostoma and Necator, because of their different pathogenic potential, but within the
genus Ancylostoma, there are two species, A. duodenale and A. ceylanicum, of which the
latter is zoonotic. The emergence of A.ceylanicum in South East Asia is a major
impediment to control where mass chemotherapy is used, because dogs are the zoonotic
reservoir of A. ceylanicum and are not targeted in mass chemotherapy programs .
Similarly, humans may be infected with more than one species of taeniid cestode in some
endemic areas where there are a variety of susceptible intermediate hosts, particularly
pigs and cattle . In such situations, the epidemiology and control of human teaniasis
and cysticercosis is dependent upon determining cycles of transmission and sources of
infection. As with hookworm, morphological discrimination of taeniid species is not
possible on the basis of the parasite stages passed in human faeces but is readily achieved
with PCR-based procedures .
In addition to providing rapid and sensitive identification of established parasite taxa, the
application of molecular tools has also helped to resolve taxonomic issues that may have
resulted in controversy in the past, when new species or ‘strains’ were described on the
basis of host occurrence, phenotypic characteristics and/or epidemiological observations.
The resolution of taxonomic issues using molecular tools often occurs in two distinct
stages. First, different genetic groups are found within what is ostensibly a single,
morphologically defined species, and then these groups are defined as taxonomic
categories, either at the intraspecific level or as different species or higher taxa.
4.1. Factors which promote genetic structure
The genetic structure (i.e. the extent to which genetic variation is distributed among,
rather than within populations) of a species is determined by the interplay of different
evolutionary forces, principally genetic drift, selection and migration. These evolutionary
forces are themselves influenced by a range of biological and ecological factors such as
mode of reproduction, breeding system, effective population size and dispersal ability.
The extent to which we recognize intraspecific groups of parasites will be a function of
the extent to which intraspecific variation is structured among different hosts or among
different geographic areas. This, in turn, will be determined primarily by the mode of
reproduction of the parasite and the fragmentation of parasite populations among host
4.1.1. Mode of reproduction
In asexual reproduction, new, genetically identical individuals are produced by a single
parent without genetic recombination. Although viruses and bacteria reproduce
predominantly asexually, they may sometimes exchange genetic material. Most parasitic
protozoa reproduce asexually, although they may also have an obligate or facultative
sexual phase in their life cycle. As a result, three different types of population structure
have been proposed for viral, bacterial and protozoan parasites: panmictic, as a result of
frequent genetic exchange; clonal, resulting from little or no genetic exchange; and
epidemic, where a basic panmictic structure is masked by occasional clonal expansion of
certain genotypes [51,52].
The extent of clonality in a species can be inferred through the pattern of single locus and
multilocus genetic diversity. At the single locus level, clonality will lead to an excess of
heterozygotes in diploid organisms, while at the multilocus level, clonality will produce
widespread, identical genotypes, non-random associations between alleles at different
loci (linkage disequilibrium) and congruence in different intraspecific gene phylogenies
[53,54,55]. A clonal population structure does not imply that genetic exchange is absent
in the species, only that it is too rare to erode the basic genetic patterns of clonality.
Using these criteria, Tibayrenc et al. [53,56] identified a number of species of parasitic
protozoa, including Entamboeba histolytica, Giardia duodenalis, Lieshmania tropica, L.
major, Trypanosoma brucei, T. cruzi and T. vivax, as having an essentially clonal
population structure. Subsequent studies have shown that the situation is rather more
complex and different populations of the same species often show different degrees of
clonality . MacLeod et al. [52,58], for example, found that Trypanosoma brucei
isolated from livestock in Botswana showed an epidemic population structure, while the
same species isolated from humans in the same locality, had a clonal population
structure. In Europe and North America, Toxoplasma gondii is considered to have a
predominantly clonal population structure, with three main clonal lineages, referred to as
Types I, II and III, accounting for >85% of strains isolated from humans and domestic
animals . However, recent studies of isolates of T. gondii in wildlife from North and
South America, as well as Australia have uncovered more biological and genetic
diversity [60,61,62]. Although some strains infecting wildlife appear to be recombinant
genotypes derived from crosses between the archetypal clonal lineages, others are
atypical strains which possess completely novel alleles. This diversity appears to be
driven by regular cycles of sexual reproduction, with occasional expansion of clonal
lineages by carnivory or self-mating [63,64]. A panmictic population structure is
therefore thought to exist in South America and parts of North America, with an
epidemic expansion of successful clones through most of North America and Europe,
where wildlife apparently plays a less significant role in transmission of the parasite
4.1.2. Fragmentation among hosts
Some parasitic protozoa and most helminths have an obligate phase of sexual
reproduction, involving genetic exchange, during their life cycle. For these parasites, the
factor of overwhelming importance in determining genetic structure is the fragmentation
of populations among hosts. The infrapopulation in a single definitive host represents the
breeding group. Eggs or larvae are passed into the external environment and/or one or
more intermediate hosts, so the progeny from different infrapopulations are mixed each
generation . The extent of mixing at different spatial scales determines the extent of
genetic differentiation among infrapopulations, among component populations in
different host species and among suprapopulations (all the individuals of a parasite
species within all hosts and in the environment) in different geographic areas. Progeny
mixing is influenced by a wide variety of intrinsic and extrinsic factors, including
transmission dynamics, asexual amplification of larval stages, inbreeding rate in the
definitive host and host migration rate.
At the level of different definitive hosts of the same species, if offspring are transmitted
vertically or as a clump from one definitive host to the next over several generations, then
infrapopulations will effectively function as demes. This is likely to promote inbreeding,
leading to the reduction of within-host genetic variation and an increase in among-host
genetic variation through genetic drift  (Fig. 1a). Evidence for a recurrence of
generations within individual infrapopulations has been found in a number of studies. For
example, infrapopulations of lice, Geomydoecus actuosi, infecting pocket gophers
(Thomomys bottae) have heterozygote deficiencies (indicating inbreeding) and are
strongly structured, with 9.2% of genetic variance distributed among hosts . Lice are
transmitted exclusively by inter host contact and in pocket gophers this principally occurs
during mating encounters and the rearing of young. Anderson et al.  found that
Ascaris worms bearing identical mtDNA haplotypes were found within the same human
or pig host more frequently than expected by chance. They suggested that this resulted
from the spatial clumping of genetically related eggs in the environment.
For many parasites, however, there is little evidence of genetic structuring among
infrapopulations, indicating that clumped transmission is rare. In populations of
Teleodorsagia from sheep, for example, 98% of genetic variation occurs within
infrapopulations , a result consistent with other studies of trichostrongyloid
nematodes [69,70]. Even in parasites which have a life cycle predisposed to self-
reinfection, such as Strongyloides ratii, less than 5% of genetic variation is distributed
among infrapopulations in definitive hosts .
The recurrence of generations within individual definitive hosts may be enhanced by
asexual reproduction in intermediate hosts, as occurs in many protozoans, digenean
trematodes and cestodes (Fig.1b). Transmission of clones from the intermediate host to
the definitive host appears to lead to enhanced structuring of infrapopulations of
Plasmodium falciparum in people  and the cestode, Fascioloides magna, in white-
tailed deer (Odocoileus virginianus) . Asexual reproduction in intermediate hosts
does not necessarily lead, however, to a stable recurrence of generations within, and
enhanced genetic diversity among definitive hosts, because it will be countered by factors
such as reduced variance in reproductive success between clones and enhanced mobility
of both definitive and intermediate hosts . Theron et al.  for example, found that
infrapopulations of Schistosoma mansoni in rats (Rattus rattus) from Guadaloupe,
contained a mean of 34 different multilocus genotypes per host despite the fact that snail
intermediate hosts (Biomphalaria glabrata) contained only 1.1 genotypes per host, on
average. The transmission of multiple genotypes to the definitive host is likely due to the
mobility of rats, their weak immune response, allowing multiple infections, and to spatial
aggregation of infected snails around limited water resources.
For parasites that are able to utilize more than one species of definitive or intermediate
host, the likelihood of genetic structuring between different component populations
depends on the extent to which the different host species utilize different resources, and
will be enhanced by the same processes that lead to structuring among infrapopulations,
that is, clump transmission and asexual multiplication. For example, Wang et al. 
identified two major genetic clusters of Schistosoma japonicum infecting different
definitive host species in Anhui province, China; one in cattle, water buffalo and humans,
and the other in goats, pigs, dogs and cats. The authors suggest that this differentiation is
due to spatial resource sharing by cattle, water buffalo and humans.
At the level of suprapopulations of parasites in different geographic areas, the major
determinant of genetic structure is host mobility, through its effect on parasite gene flow.
For example, trichostrongyloid nematode parasites of livestock (Ostertagia ostertagi,
Teleodorsagia circumcincta, Haemonchus placei and H. contortus), that are regularly
transported by people between distant locations, have less genetic structure than a related
parasite of wild deer (Mazamastrongylus odocoilei) . Similarly, ticks (Ixodes uriae)
on Atlantic puffins (Fratercula arctica) are much less genetically structured than the
same tick species on black legged kittiwakes (Rissa tridactyla); presumably because
puffins move between local colonies much more frequently than kittiwakes . For
parasites with indirect life cycles, intermediate host mobility as well as definitive host
mobility may influence genetic structure. Criscione and Blouin  found that three
species of digenean trematodes (Deropegus aspina A, Deropegus aspina B and
Plagioporus shawi) of salmonids (Oncorhynchus spp.) which cycle exclusively in aquatic
hosts, are more strongly structured than a fourth species (Nanophyetus salmincola) whose
life cycle includes highly mobile terrestrial hosts.
Host mobility can prevent geographic differentiation of parasite suprapopulations even
when parasite infrapopulations are highly structured. In the digenean trematode,
Fascioloides magna, for example, where asexual multiplication in intermediate hosts
leads to strong genetic differentiation among infrapopulations in white tailed deer
(Odocoileus virginianus), there is little differentiation among flukes in different
geographic areas, presumably because of long distance dispersal by deer . In the
cestode Echinococcus granulosus in Australia, there is no significant genetic variation
between populations from intermediate hosts in different geographic areas separated by
more than 4,000 km, despite evidence of high effective selfing rates in definitive hosts,
due to clumped transmission of clones . This lack of geographic differentiation is
presumably due to high mobility of both intermediate hosts (sheep and kangaroos) and
definitive hosts (dogs and dingoes) of Echinococcus granulosus in Australia.
4.2. Delimiting strains and species
When a substantial part of the genetic variation within a species of parasite is associated
with distinguishable biological or ecological characteristics, such as morphology, host
associations, development rate, infectivity, pathogenicity or drug resistance, we usually
wish to recognize the different variants with some formal or informal taxonomic
designation. Nomenclature is essential for effective communication and provides the
stability that underpins epidemiological investigations . The lack of morphological
differences between many inter- and intraspecific variants has, in the past, compounded
an often confusing taxonomic picture, which in many cases has taken decades to resolve.
Such was the situation with Trichinella and Echinococcus, but as a result of the
application of molecular tools many taxonomic issues have been resolved and as a
consequence, communication has been markedly enhanced. In itself, giving something a
taxonomic designation with the support of molecular data is not a ‘molecular
epidemiological’ study unless it can be put into an epidemiological context. For example,
being able to discriminate between E. histolytica and E. dispar was only possible with the
development of molecular tools which gave confidence to the species names proposed
and a terminology that underpins epidemiological investigations [81,82].
The only formal taxonomic category below the species level is the subspecies,
traditionally defined as a geographically localized intraspecific group that differs
genetically (and taxonomically) from other such groups . There are, however, a
plethora of other terms such as isolate, stock, line, strain and discrete typing unit, which
have been used informally to describe intraspecific variation (Table 2). These terms have
often been defined in different ways and in a biological sense their value is questionable
because they may tell us nothing about the evolutionary history or evolutionary potential
of the groups concerned. Their application to parasitic organisms however, has been of
great practical significance, because they are often related to important features of
parasitic disease. In Trypanosoma brucei, for example, three subspecies have historically
been defined on the basis of geographic and host distribution, and the clinical course of
disease. T. b. gambiense is a human parasite distributed through western and central
Africa, causing chronic disease. T. b. rhodesiense is a human parasite distributed through
eastern and southern Africa causing acute disease and T. b. brucei infects domestic and
game animals, but not humans and is widely distributed throughout sub-Saharan Africa.
A genetic basis to human infectivity appears to have been established, at least for some
isolates, in the expressivity of the serum-resistance-associated (SRA) gene product
[84,85]. Population genetic studies using a range of genetic markers, however, suggest
that neither T. b. gambiense or T. b. rhodesiense form monophyletic groups. It appears
that T. brucei has acquired the ability to infect humans on four separate occasions, twice
within the subspecies T. b. gambiense and twice within the subspecies T. b. rhodesiense
. Therefore, while it seems inappropriate to retain the three subspecific designations,
the three groups could still be referred to as different strains, because of differences in
geographic distribution and human infectivity.
Debate over what constitutes a species has been an enduring source of confusion in
biology, with a multitude of different species concepts proposed [86,87]. Among
parasitologists, this has frequently led to disillusionment with the prospect of identifying
a single species concept that includes all groups of parasites and the use of a purely
phenotypic definition of species [88,89]. We believe, however, that the problem posed by
this multiplicity of different species concepts can be overcome by recognizing a
fundamental distinction between conceptual views of what constitutes a species and
operational criteria for delimiting different species . Many existing species concepts,
such as the biological species concept, the phylogenetic species concept and the cohesion
species concept, differ only in their operational criteria for species delimitation;
conceptually they agree that species represent the contemporary tips of an evolutionary
lineage . They can therefore be equated with the evolutionary species concept; which
states that that a species is a single lineage of organisms with a common evolutionary
trajectory, distinguishable from other such lineages . The evolutionary species
concept is applicable to most eukaryotic organisms, regardless of their mode of
reproduction or breeding system, although it may be difficult to apply if horizontal gene
transfer is common between distant lineages . Delimiting species under an
evolutionary species concept requires a determination of when lineages have a common
evolutionary trajectory (indicating that they are the same species) or when they have
different evolutionary trajectories (indicating that they are separate species).
One approach to delimiting species is to utilise the pattern of evolutionary relationships
among lineages, such as genetic distance, monophyly or exclusivity, as a guide to their
evolutionary trajectory. Genetic distance between lineages, usually inferred with mtDNA
or ribosomal ITS markers, has frequently been used as an indicator of specific status. For
example, Mcnish et al.  suggested that isolates of Hymenolepis nana in Australia
actually exist as two cryptic or sibling (morphologically identical, but genetically
different) species, based on a sequence divergence of 5% in the mitochondrial
cytochrome c oxidase 1 gene. Such a genetic yardstick approach is a useful prospecting
tool for suggesting the possibility of different cryptic species within a morphologically
similar group, but it does not provide an infallible guide . More reliable indicators of
species status can be gained from a phylogenetic analysis of the putative species.
Organisms following the same evolutionary trajectory should be monophyletic (derived
from the same ancestral taxon) and exclusive (more closely related to each other than
they are to any individuals outside the group) . The morphologically defined species
Echinococcus granulosus, for example, has now been split into a number of different
species, because phylogenies based on mtDNA sequence data indicate that strains of E.
granulosus are not monophyletic [95,96].
Another approach to delimiting species, complementary to utilising the pattern of
evolutionary relationships among lineages, is to examine the processes, such as gene flow
and ecological constraints, which are responsible for maintaining a cohesive evolutionary
trajectory. Studies utilising this approach have typically focused on gene flow, although
that is, of course, only applicable to organisms which regularly exchange genes in their
life cycle. For parasites which can be cultured under controlled conditions, experimental
crosses can be used to determine the ability of two populations or lineages to hybridise.
Le Jambre , for example, established mixed populations of Haemonchus contortus
and H. placei in recipient sheep and found that hybrid offspring had markedly reduced
Crossing experiments, however, are not possible or practical for most parasite species and
the extent of gene flow is usually monitored in the field. Fixed genetic differences
between populations in sympatry provide strong evidence that they are on different
evolutionary trajectories and therefore represent different species. Many studies have
used genetic markers to identify such non-recombining lineages of parasites existing in
the same geographic area and often in the same host individual. The implication from
these studies is that we may have underestimated the number of independently evolving
species in almost all groups of parasites. For example, historically only two major
zoonotic species of anisakid nematodes have been recognised; the herring worm or whale
worm Anisakis simplex, and the cod worm or seal worm, Pseudoterranova decipiens,
both with an apparently cosmopolitan distribution. Recent molecular genetic studies,
however, have shown that both of these morphospecies actually comprise a number of
genetically differentiated sibling species, often with distinct geographic and/or host
ranges [98,99]. Similarly, morphological studies have identified approximately 175
species of avian blood parasites of the genera Haemoproteus and Plasmodium, but
mtDNA sequencing indicates that the real number of species may be around 10,000,
almost two orders of magnitude greater . Even in very well studied groups of
parasites, such as the trichostrongylid nematodes infecting livestock, new cryptic species
are being discovered. Grillo et al.  found that worms from a goat farm in France,
morphologically identified as Teleodorsagia circumcincta, were in fact two separate
species, with little gene flow between them.
5. Determining transmission cycles
Theoretical models of the population dynamics of micro- and macroparasites have
provided important insights into the key features of parasite transmission, particularly
with respect to the parameters that determine the basic reproduction ratio (R0) of the
parasite. Such models, however, usually make simplifying assumptions that ignore
parasite, host and environmental heterogeneity, and obtaining empirical data on how
these heterogeneities affect parasite transmission is an important requirement for
developing more realistic population dynamic models. A key empirical demand, and one
that can be addressed with molecular epidemiological tools, is to estimate networks of
parasite transmission, both within and among species of hosts. Among species of host,
most interest has centred on the application of molecular tools to infer the frequency of
zoonotic transmission in a range of parasite species.
5.1. Echinococcus, Giardia and Cryptosporidium
Species of these three parasite genera clearly share little in terms of their biology and
phylogenetic relationships. However, they all have three characteristics in common: a
wide host range and questions of host specificity; an early taxonomy poorly supported by
limited and/or questionable morphological discrimination and based largely on host
occurrence; and uncertainty about their zoonotic and public health significance .
With Echinococcus, Giardia and to some extent Cryptosporidium, molecular tools have
helped to resolve taxonomic issues and have supported the proposals of early taxonomists
[22,103,104]. Evidence of morphological differences between isolates of Echinococcus
from different hosts can now be supported by extensive molecular evidence of genetic
variation and as such can be used as reliable and cost effective diagnostic markers in field
studies, particularly in developing countries where costs and lack of equipment is an
issue. This is not the case with Giardia, where initial taxonomic descriptions based on
host occurrence could not be supported by morphological differences. Molecular tools
therefore are the only method for identification and are proving of value
epidemiologically. With Cryptosporidium, robust molecular epidemiological tools are
available but they have principally been utilised for taxonomic purposes and their full
epidemiological potential has still to be realised.
Species of Echinococcus have a two-host life cycle involving an herbivorous or
omnivorous intermediate host and a carnivorous definitive host. The parasites
demonstrate high definitive host specificity but low intermediate host specificity, which
has raised questions about the rigidity of cycles of transmission and the zoonotic potential
of populations maintained in different host assemblages . As with Giardia and
Cryptosporidium, the epidemiology of infections with Echinococcus spp., particularly in
humans (cystic or alveolar echinococcosis), was based on a species taxonomy established
largely on host occurrence. This was questioned on taxonomic grounds and in the
absence of evidence of genetic distinctness between the parasites from different
intermediate hosts, there was uncertainty for many years whether cycles involving sheep,
cattle, pigs, camels, kangaroos etc. could interact. These questions have subsequently
been resolved with the advent of reliable, robust and reproducible molecular tools which
have not only supported the early taxonomy, but also demonstrated the distinctness of
transmission cycles and the potential for interaction, particularly with respect to zoonotic
transmission [95,103,106]. Importantly, these molecular epidemiological studies have
given confidence to the morphological characters used for species discrimination which
now offer a simple, cost effective means of parasite identification in endemic foci where
the application of molecular tools may not be practical or cost-effective [107,108].
Host specificity and zoonotic potential have been the key drivers of epidemiological
investigations on species of Giardia, ubiquitous enteric protozoan parasites of mammals.
Frequent reports of infection in companion animals, livestock and aquatic mammals have
led to much discussion and controversy over their role as zoonotic reservoirs of infection
(104,109). The lack of any significant morphological variability, but considerable
evidence of phenotypic differences between isolates, fuelled this debate over many years.
The subsequent application of PCR-based molecular tools has resolved questions of host
specificity, taxonomy and zoonotic potential, but not the frequency of zoonotic
transmission [104,109]. The application of multilocus genotyping to isolates of Giardia
from human and other mammalian hosts in different parts of the world has clearly
demonstrated the occurrence of zoonotic species in the same geographic areas, supporting
the potential for zoonotic transmission [110,111]. However, finding infection with
zoonotic genotypes only demonstrates the potential for transmission – not the occurrence
of actual transmission. This is not a reflection of the tools available but of the lack of
focus in the study design, and from an epidemiological point of view, genotyping
disparate collections of isolates may not be informative.
A number of studies in defined endemic foci have provided convincing evidence of
zoonotic transmission involving dogs and humans [112,113,114]. Although the emphasis
of these studies has been on the dog as a reservoir of human infection, some recent
reports investigating the molecular epidemiology of infections with species of Giardia in
wild primates have provided further evidence of zoonotic transmission in localised foci,
and have also demonstrated that ‘reverse zoonotic transmission’ (zooanthroponotic) is an
important factor that must be considered in understanding the epidemiology of infections
with these parasites [115,116,117].
There are two zoonotic species/assemblages of Giardia which are geographically
widespread and as more isolates are genotyped, contrasting patterns are emerging of their
distribution. For example, studies in Europe had suggested that G. enterica/assemblage B
has a predominantly human distribution , but a recent study of dogs in the USA
found a higher frequency of infections with G. enterica/assemblage B than with G.
duodenalis/assemblage A . Thus in North America at least, we cannot assume that
G. duodenalis/assemblage A is the most common of the zoonotic assemblages found in
non-human hosts. Indeed in wildlife, G. enterica/assemblage B often predominates 
whereas in cattle, G. duodenalis/assemblage A is most often reported . However,
there is extensive genetic sub-structuring within G. enterica/assemblage B, and it is
possible that some subgroups are more commonly associated with zoonotic infections
than others. From an epidemiological perspective, there is increasing evidence of
differences in virulence between the zoonotic species of Giardia and how this manifests
clinically in different host species and in different circumstances associated with
nutritional deficiencies and/or polyparasitism requires much more research .
A lack of morphological differences between isolates of Cryptosporidium from different
host species, waterborne outbreaks and circumstantial evidence that livestock could have
been the source of water contamination were the main drivers for the development of
molecular epidemiological tools for these parasites. These have proved to be very useful
in determining sources of infection and risk factors of public health significance  but
have not resulted in significant epidemiological information since then. Although the
tools are available, overall, molecular epidemiological studies on Cryptosporidium
infections are considered to be still in their infancy . Molecular tools have, however,
improved our understanding of species-level taxonomy. This has largely been of value in
understanding the extent of non-human reservoirs of zoonotic infection, but with limited
application to population genetic studies. The epidemiological potential of such studies
has been demonstrated by Mallon et al.  and Peng et al. , who have provided
evidence that the population structure of C. parvum (=C. pestis ) and C. hominis is
more complex than previously suggested.
Sub-genotyping has continued to reveal genetic sub-structuring within C. parvum, but
whether this is reflected in variation in host specificity and zoonotic potential remains
unclear. Using multilocus genotyping, Grinberg et al.  provided evidence supporting
the suggestion of Hunter and Thompson  of distinct anthroponootic C. parvum
cycles that do not involve cattle. The recent demonstration of zoonotic transmission to
humans in the UK of a newly described genotype of Cryptosporidium from rabbits 
has raised concerns that humans may be at risk of infection from rabbits in other
geographic areas, as recently proposed in Australia where the rabbit genotype has been
identified . Although given a new species name, C. cuniculi, by Robinson et al.
, it is genetically very close to C. parvum and C. hominis and thus it may be prudent
to reconsider the taxonomic status of all three species in the future. Recent advances in
nucleic-based approaches for the diagnosis and analysis of genetic diversity in species of
Cryptosporidium  represent a significant step towards an improved understanding of
epidemiology and population structure .
5.2. Wildlife and zoonoses
The role of wildlife as reservoirs of infections that may be transmissible to humans is a
controversial issue. Human factors clearly play a role in increasing the risk of any
‘spillover’ of infections from wildlife through encroachment, land clearing, hunting etc.
However, there is increasing evidence of ‘reverse zoonotic’ transmission from humans to
wildlife, a factor that must be embraced if the so-called ‘one health’ concept considers
diseases of wildlife as important as those affecting humans and domestic animals. It is
important to understand parasite biodiversity in wildlife in terms of conservation
[130,131]. This requires the surveillance of fauna that may often be endangered or
threatened. In such circumstances, non-invasive sampling and the application of
molecular tools can provide data which, in the past, were only available following
opportunistic necropsy. For example, from an anthropocentric viewpoint, an
understanding of the species of Plasmodium affecting primates in the wild led to the
identification of a new zoonosis, with P. knowlesi found in humans using molecular tools
[132,133]. In contrast, the application of molecular tools has identified a novel,
genetically distinct form of Leishmania in macropod marsupials in Australia, as well as a
new non-sandfly vector [134-136]. It is likely that the ecology of both the novel form of
Leishmania and its vector have been associated with wildlife well before human
settlement of Australia, but the discovery has raised concerns about the transmission to
wildlife of introduced, pathogenic forms of Leishmania from humans or dogs .
Native rats (Rattus macleari) on Christmas Island became extinct following the
introduction of flea-infested R. rattus in the early 1900’s. It was proposed that this could
have been due to infection of naïve native rats with T. lewisi transmitted by fleas from R.
rattus [138,139]. There was no way of proving this until ancient DNA techniques were
applied to museum specimens and demonstrated the presence of T. lewisi in native rats
after colonisation of Christmas Island by R. rattus but not before . In Western
Australia, a comprehensive program of non-invasive parasite surveillance in native
wildlife has revealed a diversity of novel Trypanosoma genotypes . All are
stercorarian trypanosomes, with some closely related to the causative agent of Chagas
disease, T. cruzi. Chagas disease is no longer confined to South America because of
increasing human migration to non-endemic regions [141,142]. The occurrence of
trypanosomes related to T. cruzi in Australian native wildlife raises question about the
vectorial potential of T. cruzi in Australia, which cannot as yet be answered . As
with Leishmania sp. in Australia, the application of molecular tools will prove invaluable
in addressing these questions.
The term polyparasitism refers to concurrent/concomitant/co-/mixed infections of either
different species and/or intraspecific variants of parasites, the latter a more recently
recognised phenomenon with the advent of molecular tools. It is not a newly discovered
phenomenon as Stoll  demonstrated when estimating the huge global burden of
human nematode infections. This was greater than the global population at the time,
reflecting the large number of mixed infections in developing countries. The seminal
studies of Buck et al. [144-147] highlighted the problems of assessing morbidity due to
multiple parasite infections in highly endemic foci. Keusch and Migasena  also
emphasised that polyparasitism was the rule rather than the exception, and that the
possibility of either synergistic or antagonistic effects must be considered in planning
public health intervention programs. Yet this phenomenon has been overshadowed for
many years by the widespread use of the DALY measuring system to determine the
impact on health of parasitic disease, which does not take into consideration the “co-
morbidities of polyparasitism” . Payne and colleagues  suggest a new approach
where co-infections with more than one infectious agent are defined as a specific disease,
for example malaria, hookworm, malaria + hookworm, malaria + schistosomes, etc. This
would appear logical and should be readily achievable with diseases such as malaria and
hookworm, where the pathogenic mechanisms of the individual aetiological agents are
reasonably well understood.
Awareness of the significance of polyparasitism in terms of malaria, schistosomes and
more recently gastrointestinal helminths, is now well established [150-156]. Molecular
epidemiological tools will contribute enormously to a better understanding of
polyparasitism by providing the means to identify parasites in situations where
morphological characterisation is not possible. Probably the earliest example of this was
the demonstration of mixed infections with genetic variants of Plasmodium falciparum in
humans . The potential clinical impact of this had been demonstrated previously in
rodent models of P. chabaudi . The far reaching implications of such mixed
infections on vaccination and drug treatment strategies in malaria, with respect to
interfering with competitive interactions and promoting increased virulence, are now well
known and continue to be the subject of much debate .
In contrast, much less is known about the occurrence of genetically mixed infections of
trypanosomes . Molecular epidemiological studies have demonstrated extensive
genetic diversity in Trypanosoma cruzi in endemic regions of South America. Studies
have also revealed the wide host range of T. cruzi in terms of sylvatic cycles and vectors,
as well as the occurrence of mixed infections in reservoir hosts, which is perhaps not
unexpected given the high levels of genetic diversity which characterise T. cruzi .
Experimental studies in mice have also demonstrated that in mixed infections, strains
exhibit predilection for different tissues (histiotropism). It is therefore surprising that the
appropriate molecular tools have only recently been applied to human infections. These
have demonstrated the occurrence of mixed infections with different strains of T. cruzi
[162-164] and raised questions about the impact that differences in virulence, drug
sensitivity and histiotropism among strains of T. cruzi will have on the management of
Chagas disease . Some years ago it was suggested that T. cruzi populations in a
patient’s bloodstream could be dissimilar to the parasite population that causes tissue
damage . Using molecular tools, this has now been shown to be the case in mixed
infections where the most prevalent genotype in the bloodstream is different to that in
cardiac tissue [164,166]. This can complicate treatment involving heart transplantation,
where chemotherapy is used to counter reactivation of the parasite following post-
surgical immunosuppressive therapy, since in mixed infections parasite reactivation can
occur at different times .
The epidemiology of mixed infections with different genera of protozoa demonstrates
another situation where molecular tools provide the means to identify the causative
agents. For example, in South America, overlapping zones of transmission of T. cruzi and
Leishmania spp. occur frequently, resulting in mixed infections in humans[167,168].
Molecular tools provide a valuable alternative to traditional diagnostic approaches, which
suffer from cross-reactivity, in areas where successful treatment depends upon accurate
and speedy diagnosis. In wildlife, molecular tools have contributed to a better
understanding of the pathogenesis of infections with Toxoplasma gondii in sea otters. A
recent study has shown that in 42% of cases, sea otters infected with T. gondii were
coinfected with Sarcocystis neurona and that such mixed infections were more virulent
than single infections . Similarly, experimental studies in mice coinfected with T.
gondii and Trypanosoma lewisi demonstrated increased virulence in mixed infections
[170-172]. Molecular epidemiological studies of threatened native wildlife in Western
Australia have revealed the occurrence of mixed infections with T. gondii and
Trypanosoma spp., which may be associated with the decline of Bettongia penicillata
. Mixed infections with different genotypes of T. gondii are common in Australian
wildlife, but it is not yet known how this influences the virulence of infections .
The prevalence of mixed infections with enteric protozoa in developing countries show
that they are the rule rather than the exception, with children most at risk harbouring at
least two species of protozoan [174-178]. The most commonly represented protozoa in
concurrent infections are Giardia spp., Blastocystis spp., and Entamoeba coli, which are
usually also found with the cestode Hymenolepis nana [174-176,179,180]. Depending on
the endemic area, E. histolytica and E. dispar may also occur in mixed infections. It is not
possible to identify all these species using morphology alone and thus molecular tools
now provide the means to undertake epidemiological studies.
Multiple low-intensity infections of gastrointestinal helminths and schistosomes have
been shown to confer an increased risk of anaemia, emphasising that this common pattern
of infection is not clinically benign . Chronic helminth infections could also have a
significant influence over the immune response and hence susceptibility to other
pathogens including microparasites, both systemic and enteric, as well as influencing the
outcome of vaccine trials [182-186]. In order to determine the impact of polyparasitism in
these situations, accurate diagnosis is again important, for example in determining the
species of schistosome or hookworm involved . Molecular tools will therefore
provide the basis to better understand the epidemiology of mixed helminth and protozoan
infections. For example, the inability to identify hookworm species in the study by
Sorensen et al.  of gastrointestinal infections in Guatemalan children, leaves open
questions about the potential of zoonotic transmission, if dogs are a reservoir of A.
ceylanicum as in South East Asia (see above).
7. Markers for traits of epidemiological importance
Studies of genetic diversity may be of great practical significance not only in parasite
identification, dissecting interactions within the host and tracking transmission among
hosts, but also in inferring the distribution of genetic variation in traits of epidemiological
importance, such as drug resistance, zoonotic potential and virulence, and in predicting
the evolutionary response in such traits to selection pressures imposed by nature or by
human intervention. A number of studies using neutral allozyme or mtDNA markers, for
example, have found relatively high levels of genetic diversity within populations and
little population genetic structure in helminth macroparasites . An obvious
implication from such studies is that populations of parasites might respond rapidly to
selection imposed by drug treatment and that gene flow would rapidly spread resistance
alleles to other populations.
There is however, a limit to the usefulness of non-neutral genetic markers in
epidemiological studies. Although there are a number of examples of resistance to drugs
which appear to be largely under the control of a single gene (e.g. resistance to
chloroquine in Plasmodium falciparum  and to benzimadazole in Haemonchus
contortus ), most of the parasite traits which interest us from an epidemiological
viewpoint are likely to be polygenic, quantitative traits . A number of studies over a
wide range of taxa have shown that there is a poor correlation between the among-group
genetic variance in neutral, single gene markers (typically measured by FST) and the
among-group genetic variance in complex, quantitative traits (as measured by QST) [191-
7.1. Finding genetic markers for quantitative traits
An obvious solution to this problem of non-correspondence between FST and QST would
appear to be to utilize more non-neutral genetic markers, particularly quantitative trait
loci (QTLs), in molecular epidemiological studies. There are two common approaches to
the identification of QTLs for polygenic traits; linkage studies, which test for correlated
segregation of marker alleles and phenotypic values in families or the offspring of
experimental crosses, and association studies, which test for the correlated occurrence of
marker alleles and phenotypic values in natural populations . Either approach may
use neutral markers, which are distributed randomly throughout the genome, or candidate
markers, which are chosen because of some a priori evidence of their effect upon the trait
The dominant design for the last 20 years has been linkage analysis with neutral markers
to find regions of the genome in which putative QTLs may be located, followed by a
search for candidate loci within the mapped region. This approach has been used
successfully to map QTLs for traits of epidemiological significance in a number of
parasite species; for example drug resistance in Plasmodium falciparum , virulence
in Toxoplasma gondii  and resistance to malaria parasites in Anopheles gambiae
. Linkage analyses have been so successful because, as closely related individuals
have large portions of their genome in common, a relatively small number of
polymorphic neutral markers (usually 500 or less) are usually sufficient to detect linked
regions . They do, however, require sophisticated genetic crosses, which are often
not feasible for parasites with complex life cycles and, while they are more powerful than
association studies for detecting QTLs with large effects on a trait, they are less powerful
for detecting QTLs with small effects, because patterns of allele sharing in such cases
will be less striking between relatives than between unrelated individuals [198,199].
Association analyses have been limited in the past by the need for very dense neutral
marker coverage of the genome (typically tens or hundreds of thousands of markers) and
a paucity of suitable candidate genes. These limitations are rapidly being overcome. In
recent years, the growth of genome databases for an increasing number of parasite
species has vastly increased the number and genome coverage of SNPs and polymorphic
microsatellite loci, making genome-wide association studies much more feasible. At the
same time, the development of new genomic/proteomic tools, such as cDNA microarrays,
have greatly expanded the potential for identifying suitable candidate loci, even for
species where genome databases are not available (195,200). While these new techniques
hold much promise for the detection of QTLs for epidemiologically important traits, there
are still a number of challenges that need to be faced.
7.2. The importance of understanding population structure
Unless candidate gene markers are being used, genome-wide association analyses rely on
linkage disequilibrium (non-random association of alleles) between neutral markers and
QTLs. Linkage disequilibrium can arise, not only because of physical linkage between
loci, but also because of recent mutations, epistatic selection, genetic drift (especially in
founding populations) or the admixture of genetically differentiated populations .
Asexual reproduction and inbreeding, which limit recombination between loci, enhance
the maintenance of linkage disequilibrium and QTLs are therefore more likely to be
detected in parasite species with a clonal population structure .
For species which have regular cycles of sexual reproduction, careful selection of
populations for association studies can increase the chances of QTL detection; isolated
populations which have been recently derived from a small number of founders, for
example, should have enhanced linkage disequilibrium and would provide good targets
for an initial genome-wide QTL scan. On the other hand, an inadequate knowledge of
population genetic structure can lead to false-positive associations between markers and
QTLs. This may arise if unobserved subgroups within the population, which will differ in
allele frequencies throughout the genome, also differ in mean values for the
epidemiological trait of interest . This is of particular concern for parasites, because
subgroups are often indistinguishable morphologically and can only be detected by a
thorough population genetic study.
7.3. The importance of understanding genetic architecture
QTLs, once identified, can potentially be used to directly examine the genetic structure of
epidemiologically important traits, which will have major advantages for identifying risk
factors for parasitic disease and predicting the outcomes of treatment and control
regimes. Caution will be needed, however, in the interpretation of such data. Theoretical
analyses suggest that, unless the QTL explains a large proportion of genetic variance in
the trait, then among-group QTL variance, just like among-group neutral marker
variance, may be uncorrelated with genetic variance in the trait itself . The extent of
trait variance explained by a QTL is essentially a question of genetic architecture, i.e. of
the number, effect size and allelic interactions of all the loci affecting the trait .
A number of mapping studies have identified QTLs explaining a substantial portion of
the variance in quantitative epidemiological traits. Ferdig et al. , for example, found
that 65% of the variance in quinine resistance in Plasmodium falciparum was attributable
to allelic variation at two loci, while Behnke et al.  were able to explain 90% of the
variance in virulence between two clonal lineages of Toxoplasma gondii by allelic
variation at a single QTL. Results such as these suggest a skewed distribution of effects;
with a few QTLs with relatively large effects (major genes) influencing most of the
genetic variance, while the remainder of the genetic variance is influenced by a large
number of QTLs with much smaller effects (minor genes). This is an encouraging
finding, for it might indicate that we can concentrate on major genes and effectively
ignore the contribution from minor genes when investigating the genetic structure of
epidemiologically important traits. Unfortunately, the reality is likely to be much more
complex. First, QTL mapping studies invariably tend to underestimate the number of
QTLs affecting a trait and overestimate their effects, especially when small numbers of
progeny are analysed [205,206]. Second, mapping studies do not necessarily provide a
reliable guide to the contribution made by a QTL to the genetic variance of a quantitative
trait in natural populations, because the extent of this contribution depends critically upon
allele frequencies, which will differ among populations . Finally, there is increasing
evidence that epistatic effects (interactions among loci) explain a substantial portion the
genetic variance in many quantitative traits [203,206,208]; this means that the effect of
any particular QTL will vary depending on the genetic background.
There is, therefore, an urgent need to investigate the genetic architecture of
epidemiologically important traits in parasites. This work has hardly begun. Indeed, even
basic studies on the heritability of quantitative traits in parasites are rare, principally
because traditional quantitative genetic analyses rely on determining the covariance
structure for phenotypic resemblances between organisms with known degrees of
relatedness. Anderson et al.  show how this can be achieved without using
expensive and complicated breeding designs, by inferring relatedness from allele sharing
at microsatellite loci in natural populations of Plasmodium falciparum.
7.4. The importance of understanding the phenotype
Finding and utilising QTLs for traits of epidemiological importance will be enhanced, not
only by well designed mapping studies using the rapidly developing array of new
genomic and proteomic tools, but also by well defined and accurately measured
phenotypes . This is a particular problem for complex, emergent traits, such as
virulence, which depend on both parasite and host for their expression. Virulence is
usually defined as the parasite-induced increase in host mortality or reduction in host
fitness. While it is often regarded as a parasite trait, it should be more properly thought of
as a trait which emerges from an interaction between parasite and host [210,211].
For example, differences in virulence between the three main clonal lineages of
Toxoplasma gondii have been mapped to a small number of major genes or “intrinsic”
virulence QTLs, which encode kinases or pseudokinases found in apical secretory
organelles of the parasite [196,212,213]. The measure of virulence in all these studies
was the mortality rate of mice challenged with a standard intra-peritoneal dose of
parasites. Virulence, however, is not a static property of the genome of T. gondii and
strains of the parasite that are highly virulent in one species of host may be completely
avirulent in another host species . From the point of view of the parasite, host species
is an environmental factor that may markedly influence the phenotypic expression of
For emergent traits such as virulence, there are advantages in more carefully defining the
parasite components of the phenotype, for example traits that affect invasion of the host,
nutrient acquisition, modulation of the host cell cycle and evasion of the host immune
response. Such “intermediate” traits  may be able to be measured more precisely
and will be influenced by a smaller number of genetic and environmental factors than the
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in association studies, they should explain a greater proportion of genetic variance in the
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understanding of parasite ecology and the epidemiology of parasitic disease. A major
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The characterisation of genetic diversity in parasites (modified from ). In some cases,
there may be overlap between the tools (regions of DNA) used and function. This will
depend on the group of parasites being studied and the level of variation detectable by a
particular approach. Abbreviations: AFLP – amplified fragment length polymorphism; ITS –
internal transcribed spacer; LAMP – Loop-mediated isothermal DNA amplification; mPCR –
multiplex PCR; PCR – polymerase chain reaction; PCR-RFLP – PCR-coupled restriction
Systematics Highly conserved coding regions
e.g. SSU rDNA, certain
Moderately conserved regions
e.g. coding mitochondrial genes,
ITS rDNA, and other loci (e.g.
house-keeping genes such as
GDH, TPI, HSP, Actin, etc.); mPCR,
Variable regions e.g. allozymes,
RAPD, AFLP, PFGE, PCR-RFLP,
pyrosequencing, mPCR, LAMP,
Systematics / diagnosis /
Population genetics / breeding
systems (e.g. cross vs self
fertilisation) / host specificity /
molecular epidemiology /
conservation (e.g. predicting
susceptibility to pathogens) /
biosecurity (exotic and emerging
‘Fingerprinting’ / Molecular
epidemiology – tracking
transmission of subgenotypes /
sources of infection and risk
factors / competitive interactions
and course of infection
Identifying phenotypic traits of
clinical and epidemiological
significance, e.g. virulence,
infectivity, drug sensitivity
individual isolates /
clonal lineages /
Genetic markers /
linking phenotype and
Fingerprinting techniques e.g.
Mini / microsatellites, SSCP,
Genotype linked to phenotype
via i) genetic map; ii) RDA; iii)
sequencing and / or RT PCR of
genes thought to be linked to
Definitions of the main intraspecific taxonomic terms used for parasites, adapted from
Isolate An intraspecific group of (typically asexually) reproducing
microparasites (viruses, bacteria, protozoa) or larval trematodes or
cestodes, that have been obtained from a particular host individual
at a particular time. Does not necessarily imply a clonal group.
Stock An isolate that has been cultured in the laboratory for some time.
Line A subgroup of a reproducing isolate which is genetically and
phenotypically distinct, and therefore represents a single, clonal
Strain An intraspecific group of parasites that differs genetically from other
such groups in one or more traits of relevance to the treatment or
control of parasitic disease
unit (DTU) (clade)
Similar to strain, but generally reserved for a monophyletic group
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Fig 1. Factors which promote recurrence of generations and therefore genetic structuring
among infrapopulations of parasites.
(a) Clumped transmission of infective stages released into the environment. (b) Asexual
multiplication of larvae in intermediate host. Open triangles represent definitive hosts,
open rectangles represent intermediate hosts. Closed circles and diamonds are parasites
from different infrapopulations. Adapted from .