Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98 (Suppl.

I): 39-46, 2003 39

Evolution of Ascariasis in Humans and Pigs: a Multi-disciplinary

Approach
Odile Loreille/+, Franoise Bouchet*
Institute of Evolutionary and Ecological Sciences, University of Leiden, PO Box 9516, 2300 RA , Leiden, the Netherlands
*Laboratoire de Paloparasitologie CNRS ESA 8045, Universit de Reims, Reims, France

The nematode parasite Ascaris lumbricoides infects the digestive tracts of over 1.4 billion people worldwide, and
its sister species, Ascaris suum, has infected a countless number of domesticated and feral pigs. It is generally
thought that the putative ancestor to these worms infected either humans or pigs, but with the advent of domestica-
tion, they had ample opportunity to jump to a new host and subsequently specialize and evolve into a new species.
While nuclear DNA markers decisively separate the two populations, mitochondrial sequences reveal that three
major haplotypes are found in A. suum and in A. lumbricoides, indicating either occasional hybridization, causing
introgression of gene trees, or retention of polymorphism dating back to the original ancestral species. This article
provides an illustration of the combined contribution of parasitology, archaeoparasitology, genetics and
paleogenetics to the history of ascariasis. We specifically investigate the molecular history of ascariasis in humans
by sequencing DNA from the eggs of Ascaris found among ancient archeological remains. The findings of this
paleogenetic survey will explain whether the three mitochondrial haplotypes result from recent hybridization and
introgression, due to intensive human-pig interaction, or whether their co-occurrence predates pig husbandry,
perhaps dating back to the common ancestor. We hope to show how human-pig interaction has shaped the recent
evolutionary history of this disease, perhaps revealing the identity of the ancestral host.
Key words: Ascaris - Ancient DNA - archaeoparasitology - Ascaris suum - Ascaris lumbricoides

Ascariasis is a disease caused by the nematode As- and A. lumbricoides share similar migratory and develop-
caris lumbricoides and is one of the most widespread ment patterns in their hosts. In fact, there has been much
parasitic infections in humans, affecting over 1,47 billion speculation over the years as to whether or not A.
people worldwide, particularly in tropical and subtropical lumbricoides Linnaeus, 1758 from humans and A. suum
areas (Chan et al. 1994, Crompton 1999). The life cycle is Goeze, 1782 from pigs were originally the same parasite
direct, which means that no intermediate hosts are in- that evolved into two separate species. Today, they con-
volved. These worms are mainly present in communities tinue to be discussed as if they were separate species,
where poverty prevails, where there is a lack of safe drink- although it is very difficult to distinguish them. Generally,
ing water and where sanitation is poor, but A. lumbricoides the adults can be identified based on morphological char-
is also present in the USA, where about four million people acters but there is still no convenient method for discrimi-
are infected (Valentine et al. 2001) as well as in Europe nating between an egg of A. lumbricoides and A. suum.
(Muller 1975, Denham 1984, Nikolic et al. 1998, Tomaso et Numerous studies have attempted to clarify their status
al. 2001). In Austria for example, ascariasis is the third using morphological (Sprent 1952, Ansel & Thibaut 1973),
most prevalent helminthic infestation (Tomaso et al. 2001). immunological (Kurimoto 1974, Nadler 1987), karyotypic
A closely related sister species of A. lumbricoides (He et al. 1986), and biochemical approaches (Mikulikova
infects pigs. Goeze described it for the first time in 1782 1976, Paggi et al. 1985), but results were difficult to inter-
and called it A. suum. The swine worm is responsible for pret because most worms were obtained from different
significant economic losses: it reduces production effi- locations, rendering the differentiation between geo-
ciency and causes organ condemnations at slaughter due graphic and host-related variation impossible.
to pathology produced by parasite larval stages. Should A. suum and A. lumbricoides be regarded as
For many years, parasitologists thought that the life two different species as it is currently the case? Today
cycle of A. suum was identical to that of the human para- there are six different species concepts in use, all with
site, A. lumbricoides (Fig. 1). However, Murrell et al. (1997) strengths and weaknesses, and this makes it very diffi-
recently demonstrated that A. suum L2 larvae in the pig cult, if not impossible, to settle the status of A. suum and
host have a stronger predilection for the caecum and co- A. lumbricoides once and for all. In order to know if the
lon intestinal mucosa than for the small intestine. This two worms are the same or separate species in BSC
finding raises important questions as to whether A. suum biological species concept (Dobzhansky 1937) it is im-
portant to determine whether worms from one sex retrieved
from pigs and worms from the other sex retrieved from
humans can mate and produce viable offspring. The re-
sults of this hybridization experiment would require the
+Corresponding author. E-mail: [email protected] production of eggs which give rise to successive genera-
Received 26 August 2002 tions of fertile worms thereby establishing in biological
Accepted 25 November 2002 terms that A. lumbricoides and A. suum are the same spe-
40 Ascariasis in Humans and Pigs O Loreille, F Bouchet

2 3 Crewe & Smith 1971, Lord & Bullock 1982) and that pigs
may be infected with parasite ova from humans (Galvin
1968). Although these experiments are very important, until
recently they were not considered very reliable because
the immune mechanism of the pigs was distorting the re-
sults of the experiments (in some circumstances pigs have
the ability to self-cure). The other problem is that the large
doses of ova used in these experimental infections may
be unrepresentative of the natural transmission of Ascaris.
Fortunately, in 1996, a new and promising method for trans-
ferring adult Ascaris was described by Jungersen and the
4 outcome of future experiments in this area are awaited
1
with great interest (Jungersen et al. 1996, 1997).
In natural conditions, Anderson (1995) showed that
in North America, a pig ascarid infection could occasion-
ally be spread to humans and that it mostly occurred when
infective eggs found in the soil and manure were ingested.
Infection is more likely if pig feces are used as fertilizer in
5 gardens and crops become contaminated with ascarid eggs.
6 However, these natural cross-infection events seem to be
quite limited. In fact, Anderson showed in another study
that, in Guatemalan villages, parasites infecting humans
and parasites infecting pigs were reproductively isolated
(Anderson et al. 1993, Anderson & Jaenike 1997). By
Fig 1: Ascaris suum life cycle, (from Jungersen 1998). 1: ingestion studying the mitochondrial diversity from several popu-
of infective eggs; 2: the larvae migrate up the bronchi and are lations of pig and human Ascaris, he concluded that there
swallowed; 3: the larvae establish in the small intestine and become was little or no gene flow between the two populations of
patent at week 6-8; 4: hatched larvae penetrate the large intestinal worms and he ruled out the hypothesis stating that As-
wall and migrate via the liver to the lungs; 5: unembryonated egg in
faeces; 6: infective egg with L2 larva.
caris had a single transmission cycle in which humans
and pigs are alternative hosts (Anderson 2001). At least
two hypotheses could explain the discrepancy found be-
tween the North American and the Guatemalan data: ei-
cies (Crompton 2001). Although this area of research is ther cross infections do occur in Guatemala but since they
active, so far no significant result has been obtained are rare, they have been detected with the molecular mark-
(Jungersen pers. commun.). ers used by Anderson, or there is a difference in host
For the past ten years, several disciplines, offering immunity between populations.
different perspectives for elucidating the history of the With an estimated 8,000 to 100,000 children dying from
Ascaris, have extensively progressed. We will now re- ascariasis every year (Editorial 1989), it is crucial for the
view the inferences from parasitology, archeoparasitology, World Health Organization to know more about those
genetics and paleogenetics since it became obvious that worms to develop and promote efficient drugs and con-
a interdisciplinary approach will obviously be the best trol strategies. If it turns out that pig Ascaris can infect
way to understand the history of Ascaris. humans (and vice versa), there seems to be little point in
INFERENCES FROM PARASITOLOGY spending resources on anthelminthic drugs because re-
infection would seem inevitable, particularly in farms
Regarding the fidelity of the parasite to its host, it is where human populations use contaminated pig manure.
crucial to determine if the two Ascaris have a single trans- It is also important to determine if species other than pigs
mission cycle in which humans and pigs are alternative or humans can serve as reservoir hosts. A. lumbricoides
hosts or if they have two sympatric life cycles, with one might have been identified in squirrels (Rausch & Tiner
using humans as a host and the other the pigs. 1948), but the authors doubted that the very large worms
In his thesis, Jungersen (1998) reports an experiment could mature in such small animals. Further evidence sug-
performed by Buckley (1931), who deliberately ingested a gests that A. lumbricoides can also infect bears (Juniper
piece of bread contaminated with twenty larvae collected 1978) and primates (Stuart et al. 1990), and that acciden-
from the lung of a pig recently infected with a massive tally A. suum could infect cattle and sheep.
dose of A. suum eggs. Simultaneously, Buckley infected a
green monkey and two pigs with a much bigger amount of INFERENCES FROM ARCHEOPARASITOLOGY
larvae. Three months after the transfer, Buckley and the Archeoparasitology appeared as a new branch of para-
green monkey still had not shown any clear sign of infec- sitology in the beginning of the 20th century, when a new
tion whereas both pigs were found to harbor large quan- technique to rehydrate desiccated tissues allowed the
tities of adult worms. However, new cross infection ex- finding of Schistosoma haematobium eggs in infected
periments have demonstrated that humans can actually kidneys of 3,200 year-old Egyptian mummies (Ruffer 1910).
be infected with eggs derived from pigs (Takata 1951, The other main sources for parasite remains are the co-
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98 (Suppl. I), 2003 41

prolites that are desiccated or mineralized faecal material are infected by A. suum, so it is possible that ancient popu-
found at archaeological excavations. Today human-para- lations of wild pigs were infected by Ascaris before be-
site coevolutionary studies based on archeoparasitologi- coming domesticated. If this hypothesis is true, A. suum
cal findings are widely performed, showing the antiquity should be the only roundworm present in deposits pre-
of parasite-host relationships and tracing the origin and ceding pig domestication. To investigate the interactions
dispersion of the infection to prehistoric times (Araujo & between Ascaris, pigs and humans, it is thus crucial to
Ferreira 2000). use a dynamic multifaceted approach involving the his-
Parasitologists have reported that Ascaris eggs are tory of our domestic pigs.
difficult to exterminate. They remain viable in soil for as Ancestors of todays swine date back to the Miocene.
long as 15 years and are almost as widespread in the envi- The hunting of wild pigs by early humans was often de-
ronment as bacteria (OLorcain & Holland 2000). picted in Western European cave and rock paintings as
Archeoparasitologists have also reported that fossilized early as 25,000 years ago. Domestic pigs all descend from
Ascaris eggs were common in archaeological sites. Unlike a single species, the Eurasian wild boar, Sus scrofa (Epstein
bacterial or viral pathogens, nematodes produce eggs that & Mason 1984). Their domestication probably occurred
are easy to detect under a microscope and which can con- over 9,000 years ago in the Middle East and eastern Medi-
firm the presence of an infectious disease even if the re- terranean although an alternative view places the begin-
mains of the host are poorly preserved or absent. The ning of swine domestication in Southeast Asia. Sauer sug-
relative abundance of eggs on archaeological sites can gested that the pig diffused from there northward to China
be attributed to their thick shell which seems to improve (Sauer 1952). Today most archaeologists favor the hy-
their preservation through time (Fig. 2). Furthermore, the pothesis that two separate pig domestications occurred
fact that adult females Ascaris females lay on average at different times and places in the Old World. Domesti-
200,000 eggs per day increases significantly the chances cated pigs then progressively spread across Asia, Eu-
of recovering fossilized nematodes. rope and Africa.
If wild pigs are the ancestral hosts of Ascaris and if
the host shift really occurred during pig domestication,
then archaeoparasitologists should not find fossilized A.
lumbricoides in deposits preceding the arrival of domes-
ticated pigs.
The oldest Ascaris eggs recovered so far were col-
lected in the 30,000 year-old upper-Paleolithic site of
Arcy-sur-Cure, Yonne, France (Bouchet et al. 1996). This
cave is characterized by several wall paintings and by the
large amount of Ursus spelaeus remains. No pig remains
were identified. The authors stated that even if wild pigs
had been present close to the site, they would not have
gone so far inside the cave and thus concluded that the
Ascaris samples were most probably A. lumbricoides eggs.
If A. lumbricoides were present in France before the ar-
rival of domesticated pigs, it seems unlikely that domesti-
Fig 2: ancient egg of Ascaris sp. with a broken shell after passage of
ultrasonic wave (20 min at 60C). cated pigs infected humans with Ascaris. In contrast, we
can suggest that hominids were in fact the ancestral hosts
and that they later infected their domesticated pigs. Fi-
nally, since pigs ingest human feces (and not vice versa)
the hypothesis stating that humans were the ancestral
The presence of Ascaris in Europe, Africa, and even hosts is very plausible.
in the New World indicates that Ascaris represents a very To check this hypothesis, we will analyze more fossil-
successful human nematode. It evolved mechanisms that ized Ascaris eggs. Although, on a morphological basis
promoted infection in diffuse populations living in ex- only, it is impossible to determine if a fossilized Ascaris
treme and cold environments. All these findings suggest egg belongs to A. suum or to A. lumbricoides, sometimes,
that humans and Ascaris have a very long history. An- like in Arcy-sur-Cure, circumstances around the discov-
cient Greeks and Romans were familiar with Ascarids, and ery can help. For example, here we report evidence of the
intestinal complaints were mentioned in the Ebers Papy- presence of ascariasis in past populations of Nubia, in
rus and in writings of Chinese medicine. Africa. The Nubians inhabited the area along the Nile from
Although A. suum and A. lumbricoides eggs cannot the first cataract south to the sixth cataract. Today this
be morphologically distinguished, archeoparasitology land lies inside of Sudan. It was known as the Kingdom of
might be a very promising discipline for the identification Kush and it even ruled over Egypt for a while. Sa, one of
of the ancestral hosts of Ascaris. Were the first hosts the largest islands in the Nubian Nile, includes a large
humans or pigs? The actual hypothesis states that A. number of archaeological sites ranging from Palaeolithic
lumbricoides evolved from a pig Ascaris, presumably A. to more recent times (Geus 1995). Because of the regions
suum, following the domestication of pigs by humans in very arid climate, many naturally mummified remains have
the Neolithic or Paleolithic (Kliks 1983). Today wild pigs been found on the island. Very well preserved Ascaris
42 Ascariasis in Humans and Pigs O Loreille, F Bouchet

egg samples were recovered inside at least two human data is being used to infer ancestral relationships among
mummies, one dated between 300-1500 AD, and the other pathogens and to help address relevant questions about
dated between 2050-1750 BC. The context of the finding their origins. The evolution of nematode mtDNA is gen-
makes it almost certain that the samples belong to the A. erally quite unusual: it is highly A+T rich, prone to recom-
lumbricoides species. bination and gene rearrangement, and evolves faster than
In order to know more about the history and migration mitochondrial DNA (mtDNA) of other taxa (Blouin et al.
of the two worms, it is not only important to determine the 1998). Although the complete mtDNA genome of A. suum
ancestral host but also crucial to learn more about cross has been sequenced, there is still little data about the
infections. One can suppose that if cross infection can genetic diversity of both Ascaris species. Several com-
occur today, they were probably very rare when humans parative studies are needed, using similar sampling de-
and pigs had little or no contacts. Thus, it would be inter- signs and the same DNA markers, including nuclear loci
esting to search for fossilized Ascaris in areas where pigs and further work with mtDNA.
have been introduced recently. For example, one interest- In studies involving Ascaris from several localities,
ing location to search for samples of ancient A. differences in DNA patterns have been used to differen-
lumbricoides samples is the New World. tiate the two parasites. Anderson et al. (1993) analyzed
For many years scientists thought that the prehistoric mitochondrial markers to investigate the transmission of
New World was free of parasitic disease. This belief was Ascaris from two sites in Guatemala. The sequences they
then discredited in the late 1970s by archeoparasitologists obtained generally fell into two distinct clusters that are
who showed that parasites maintained infections as hu- 3-4% divergent: cluster A predominantly identified in para-
mans migrated through the Bering Land Bridge into the sites from pigs, and cluster B in parasites from humans.
New World. Patrucco reported evidence of infertile eggs The authors concluded that Ascaris from pigs and hu-
of Ascaris in coprolites collected in the site of Los mans have two distinct transmission cycles with little or
Gavilanes, Peru, and dated 2277 BC (Patrucco et al. 1983). no gene flow between populations. However, surprisingly,
Reinhard et al. (1987) identified Ascaris in coprolites from they noticed that some mitochondrial haplotypes were
five archaeological sites from the Colorado Plateau of present in worms obtained from both host-species. The
Arizona and Faulkner encountered the A. lumbricoides in authors presented the following hypotheses to explain
eight samples of desiccated human faeces from Big Bone their data: (1) the cross-infection hypothesis: A. suum and
Cave, Tennessee (Faulkner et al. 1989). Associated C 14 A. lumbricoides represent two reproductively isolated
dated material suggested an age of 2177 145 BC. As- parasite populations specializing on two different hosts
caris eggs were also identified in the abdominal cavity of and defined accurately by both nuclear and mtDNA, but
a child found at Zero Point, Alaska, and dated 840 40 BP they show incomplete host fidelity, or (2) the evolution-
(Bouchet et al. 2001). In this context, the authors also ary hypotheses: either low levels of gene flow led to in-
concluded that the parasites were undoubtedly A. trogression of genes between host-associated parasite
lumbricoides. populations (introgression), either the separation between
The oldest human remains recovered so far in the New A. suum and A. lumbricoides is too recent for alleles to
World come from the site of Monte Verde and are most have reached a state of reciprocal monophyly (retention
likely descendants of people who reached North America of ancestral polymorphism).
by crossing the Bering land bridge from Asia at least 12,000 To verify these hypotheses, Anderson and Jaenike
years ago, perhaps more (Greenberg et al. 1986). In con- (1997) compared patterns of variation in the mitochon-
trast, the introduction of swine in the New World occurred drial and in the nuclear genome of Ascaris using the para-
recently, during Christopher Columbus second voyage sites they had studied and 42 additional parasites from
in 1493 (Gade 2000). From an original stock of eight, pigs worldwide locations. A concordance between nuclear and
multiplied on the Caribbean island of Hispaniola. Later, mitochondrial markers would support the cross-infection
13 pigs arrived in what is now the United States from hypothesis, while a lack of such support would favor the
Cuba with Hernando de Sotos expedition (1539-1542) alternative hypotheses. The results obtained with mul-
through the Atlantic Coast of Florida. The explorer of- tiple nuclear markers were incongruent with that revealed
fered live pigs to the Indians, and when he died in 1542 by mtDNA. The authors concluded that cross-infection
pigs then amounted to over 700, not including those that is extremely rare and/or very short duration. In this new
had wandered away from the constraints of domestica- study three major clades (A, B, C), which differ in se-
tion to reach the vast American wilderness. The fossil- quence by up to 5.8%, were identified (Fig. 3). Despite
ized Ascaris samples identified in the New World (Patrucco lacking a solid calibration for the Ascaris molecular clock,
et al. 1983, Reinhard et al. 1987, Faulkner et al. 1989, Bouchet the authors estimated divergence among the three major
et al. 2001) are certainly the descendants of worms brought clades, and cautiously concluded that their data favored
by colonists. Unless these people were accidentally in- retention of ancestral polymorphisms over introgressive
fected by A. suum, it is very probable that A. lumbricoides hybridization (Fig. 4). Although they also concluded that
entered the New World long before A. suum. their data was favoring a single host shift, they were un-
INFERENCES FROM GENETICS able to determine who the ancestral host of Ascaris was.
One would expect that parasites infecting the ancestral
Genetic typing of modern pathogenic agents has gone host will be more genetically diverse than those that re-
through impressive developments in the past 10 years, cently colonized a new host species, but among the seven
thanks to advancements in molecular biology. Sequence polymorphic nuclear loci examined by the authors, 22 al-
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98 (Suppl. I), 2003 43

leles were found in worms from pigs and 20 in worms from tool to the discernment of infectious diseases in the past.
humans. This slightly greater genetic diversity in worms For example, Trypanosoma cruzi DNA was recovered us-
from pigs could suggest that pigs served as the ancestral ing PCR in Chilean human mummified remains (Guhl et al.
host and would thus contradict the fossil record. How- 1999, Ferreira et al. 2000). Those two studies enabled the
ever, the authors admit that the analysis of more loci is identification of Chagas disease in ancient bodies in the
necessary to better resolve the relationships between the absence of recognizable anatomic pathological changes.
two worms and to determine the region in which the host However, the retrieval of ancient DNA sequences is
shift occurred. far from routine and since nobody had ever successfully
INFERENCES FROM PALEOGENETICS amplified ancient DNA from helminthes before, we started
our investigation with a preliminary study to determine
Molecular biologists use molecule patterns to infer whether we could extract and amplify DNA from embryos
relationships between organisms. Unfortunately, the in- preserved in ancient parasite eggs (Loreille et al. 2001).
creasing interconnections among populations often make The Middle-Age site of Place dArmes in Namur (Bel-
data difficult to interpret. For example, in the case of As- gium) was selected because of its excellent taphonomic
caris, it is possible that the host shift occurred during the conditions (permanently humid and anaerobic). Several
Neolithic because pigs started to be domesticated by coprolites were recovered from a 14th Century latrine that
human populations with a size and a density important also contained abundant archaeological material. The
enough to maintain sustained epidemics. Then, during coprolites revealed a very high concentration of parasite
the next thousand years, due to the increasing mobility of eggs, some of which still had fossilized embryos inside.
humans, parasites previously confined to specific geo- Based on their morphology, we identified the samples as
graphical locations were able to spread widely. Ascaris sp. eggs, although it could not be determined if
In the 1980s, it was shown that molecules of DNA they belonged to A. suum or to A. lumbricoides. We suc-
could be preserved in ancient soft tissues (Higuchi et al. cessfully extracted DNA from 104 fossilized eggs. A 176
1984, Pbo 1985) and in bone material (Horai et al. 1989, base-pair fragment of nuclear 18S ribosomal RNA and a
Hagelberg et al. 1989). Today, by using the polymerase 98 base-pair fragment of the mitochondrial gene cyto-
chain reaction (PCR), it is possible to detect and amplify chrome b were amplified without contamination. These
the rare DNA fragments that have been scarcely preserved sequences confirmed the morphological identification to
in fossils, including DNA from the pathogens they hosted. Ascaris and proved that our sequencing techniques could
Using this approach, the spectrum of infectious diseases work on ancient samples. Unfortunately it was difficult
that can be studied in ancient samples has been consider- from our results to identify the species of Ascaris that
ably enlarged and it is now possible to get a precise diag- was present in the archaeological site. Currently we are
nostic even before any clear sign of infection, and to dis- trying to amplify more loci in order to determine if those
tinguish between pathologies and ante mortem lesions. eggs belong to A. suum or to A. lumbricoides. In addition
Thus, molecular paleopathology constitutes a powerful

P
P

P
P
H

P P

H H

P
H H

Fig 3: global distribution of the three major clades of mitochondria DNA (mtDNA) in Ascaris populations from world-wide locations (from
Anderson & Jaenike 1997). Each pie chart represents a single parasite population: those marked with a P were obtained from pigs, while
those marked H were from humans. Where parasites have been obtained from both host species from the same location (Guatemala and
Peru) two pie charts are connected with a line. The insert (bottom left) shows the phylogenetic relationships between the three mtDNA
clades. Sample sizes for parasites from pigs were: Guatemala, 38+36; Peru, 11; Switzerland, 19; Scotland, 12; USA, 20; Philippines, 18.
Sample sizes for parasites from humans were: Guatemala, 39+42; Peru, 14; Madagascar, 15; and Bangladesh, 18.
44 Ascariasis in Humans and Pigs O Loreille, F Bouchet

(1) chondrial differentiation. We can also suggest that pre-

Colombian Amerindians would have been at least par-
tially immune to the introgressive effects of hybridization
between the parasites of pigs and pig farmers in the Old
World. Hypotheses I and II both propose that pig domes-
tication coincides with the parasite jumping to a new host
and subsequently becoming a new species.
Hypothesis III suggests that the host switch and the
mitochondrial differentiation coincided, but both are an-
cient in origin and neither is the result of pig domestica-
tion. The main effect of domestication was to mix up mito-
chondrial lineages through hybridization.
Thus, hypotheses II and III propose that haplotype
(2) differentiation coincides with speciation, but occasional
cross-infection and hybridization following the advent of
domestication causes interspecific xenology of mitochon-
dria, with A haplotypes jumping into the human parasite
and B haplotypes jumping into the pig parasite. Presum-
ably, this hybridization is sufficiently common to con-
taminate the mitochondrial pattern, but insufficient to dis-
turb the two distinct clusters of nuclear RFLP markers.
Anderson favors hypothesis I because the haplotypes
seem too divergent to have coincided with pig domesti-
(3) cation, which occurred only 9 to 15,000 years ago. How-
ever, without an Ascaris molecular clock, it is hard to evalu-
ate this claim. Even if the haplotypes are ancient, they
may well have coincided with a host shift as in hypoth-
esis III, but this would have occurred between early hu-
mans and non-domesticated wild boars.
Sequence analysis of nuclear and mitochondrial mark-
ers will show whether ancient samples cluster among
modern ones, or whether they cluster together closer to
the root of the human (or pig) parasite clade, suggesting
A, B and C that even relatively recent time has led to continuing di-
mtDNA vergence between the genomes of the pig and human
parasites.
CONCLUSION
Fig 4: a possible historical scenario for the peculiar distribution of
mitochondria DNA (mtDNA) haplotypes in Ascaris, based on re- Paleogenetics may thus be developed as a new effi-
tained ancestral polymorphisms (from Anderson 2001). A three- cient tool to parasite evolution studies. DNA sequences
stage process is envisaged: (1) geographical isolation of ancestral from ancient samples should help determine the rhythm
Ascaris populations leading to the evolution of divergent mtDNA
haplotypes (including clusters A, B and C observed today); (2) 10- of evolution of Ascaris, the migration history of the popu-
2,000 years ago-mixing of Ascaris populations bearing divergent lations and hopefully the ancestral host. In conclusion,
mtDNA; and (3) host specialization leading to current situation in a close collaboration between archaeologists, parasitolo-
which A, B and C clusters of mtDNA are found in populations of gists, archaeoparasitolgists, geneticists and paleogeneti-
parasites infecting both humans and pigs. This scenario is based on
global mixing of a previously isolated parasite population, as a
cists will certainly shed light on the overall history of
result of domestication and recent human migrations. ascariasis in humans and in pigs.
ACKNOWLEDGEMENTS
To Dr Klaas Vrieling, Dr William Piel, and Mrs Milka
we plan to amplify and sequence ancient DNA from New Pejovic-Roller for critical reading of the manuscript and fruitful
World and more Old World Ascaris samples to tweeze comments.
apart the three following hypotheses: REFERENCES
Hypothesis I suggests that mitochondrial differentia-
Anderson TJC 1995. Ascaris infections in humans from North
tion predates the human-pig host switch. This ancestral
America: molecular evidence for cross-infection. Parasitol-
polymorphism is retained in the descendant species and ogy 110: 215-219.
therefore all three haplotypes should be detectable among Anderson TJC 2001. The dangers of using single locus markers
all descendant groups, including those infecting pre-Co- in parasite epidemiology: Ascaris as a case study. Trends
lombian Amerindians that did not domesticate pigs. Parasitol 17: 183-188.
Hypothesis II suggests that the human-pig host Anderson TJC, Jaenike J 1997. Host specificity, evolutionary
switch is due to pig domestication and triggered mito- relationships and macrogeographic differentiation among
 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 98 (Suppl. I), 2003 45

Ascaris populations from humans and pigs. Parasitology Higuchi RG, Bowman B, Freiberger M, Ryder OA, Wilson AC
115: 325-342. 1984. DNA sequences from the quagga, an extinct member
Anderson TJC, Romero-Abal ME, Jaenike J 1993. Genetic struc- of the horse family. Nature 312: 282-284.
ture and epidemiology of Ascaris populations: patterns of Horai S, Hayasaka K, Murayama K, Wate N, Koike H, Nakai N
host affiliation in Guatemala. Parasitology 107: 319-334. 1989. DNA amplification from ancient human skeletal re-
Ansel M, Thibaut M 1973. Value of the specific distinction mains and their sequence analysis. Proc Japan Acad serB
between Ascaris lumbricoides Linn 1758 and Ascaris suum 65: 229-233.
Goeze 1782. Int J Parasitol 3: 317-319. Jungersen G 1998. Experimental Ascaris suum infections, The-
Araujo A, Ferreira LF 2000. Paleoparasitology and the antiq- sis, The Royal Veterinary and Agricultural University,
uity of human host-parasite relationships. Mem Inst Copenhagen, Denmark.
Oswaldo Cruz 95 (Suppl. I): 89-93. Jungersen G, Eriksen L, Nansen P, Fagerholm HP 1997. Sex-
Blouin MS, Yowell CA, Courtney CH, Dame JB 1998. Substi- manipulated Ascaris suum infections in pigs: implications
tution bias, rapid saturation, and the use of mtDNA for for reproduction. Parasitology 115: 439-442.
nematode systematics. Mol Biol Evol 15: 1719-1727. Jungersen G, Eriksen L, Nielsen CG, Roepstorff A, Nansen P
Bouchet F, Baffier D, Girard M, Morel P, Paicheler JC, David 1996. Experimental transfer of Ascaris suum from donor
F 1996. Paloparasitologie en contexte Plistocne: pre- pigs to helminth naive pigs. J Parasitol 82: 752-756.
mire observations la Grande Grotte dArcy-sur-Cure Juniper I 1978. Morphology, diet and parasitism in Quebec
(Yonne), France. C R Acad Sci III 319: 147-151. black bears. Canadian Field-Naturalist 92: 186-189.
Bouchet F, West D, Lefevre C, Corbett D 2001. Identification Kliks MM 1983. Paleoparasitology: on the origins and impacts
of parasitoses in a child burial from Adak Island (Central of human-helminth relationships. In NA Croll, JA Cross
Aleutian Islands, Alaska). C R Acad Sci III 324: 123-127. (eds), Human Ecology and Infectious Diseases, Academic
Buckley JJC 1931. An observation on human resistance to in- Press, New York, p. 291-309.
fection with Ascaris from the pig. J Helminthol 9: 45-46. Kurimoto H 1974. Morphological, biochemical and immuno-
Chan MS, Medley GF, Jamison D, Bundy DA 1994. The evalu- logical studies on Ascaris lumbricoides Linnaeus, 1758 and
ation of potential global morbidity attributable to intestinal Ascaris suum Goeze, 1782. Jpn J Parasitol 23: 251-267.
nematode infections. Parasitology 109: 373-387. Lord WD, Bullock WL 1982. Swine Ascaris in humans. N Engl
Crewe W, Smith DH 1971. Human infection with pig Ascaris J Med 306: 113.
(A. suum). Ann Trop Med Parasitol 65: 85. Loreille O, Roumat E, Verneau O, Bouchet F, Hnni C 2001.
Crompton DW 1999. How much human helminthiasis is there Ancient DNA from Ascaris: extraction amplification and
in the world? J Parasitol 85: 397-403. sequences from eggs collected in coprolites. Int J Parasitol
Crompton DW 2001. Ascaris and ascariasis. Adv Parasitol 48: 31: 1101-1106.
285-375. Mikulikova L 1976. Studies on species specificity of proteins
Denham DA 1984. Ascaris lumbricoides in English schoolchil- in Ascaris lumbricoides and Ascaris suum. Folia Parasitol
dren. Trans R Soc Trop Med Hyg 78: 566-567. (Praha) 23: 45-50.
Dobzhansky T 1937. Genetics and the Origin of Species, Co- Muller WA 1975. Infestation with intestinal helminths in the
lumbia University Press, New York. inhabitants of the city of Dresden. Z Gesamte Inn Med 30:
Editorial 1989. Ascariasis. Lancet 1 (8645): 997-998. 239-247.
Epstein H, Mason IL 1984. Evolution of Domesticated Ani- Murrell KD, Eriksen L, Nansen P, Slotved HC, Rasmussen T
mals, Longman, London and New York, chapter 2, p. 6-27. 1997. Ascaris suum: a revision of its early migratory path
Faulkner CT, Patton S, Johnson SS 1989. Prehistoric parasit- and implications for human ascariasis. J Parasitol 83: 255-
ism in Tennessee: evidence from the analysis of desiccated 260.
fecal material collected from Big Bone Cave, Van Buren Nadler SA 1987. Biochemical and immunological systematics
County, Tennessee. J Parasitol 75: 461-463. of some ascaridoid nematodes: genetic divergence between
Ferreira LF, Britto C, Cardoso MA, Fernandes O, Reinhard K, congeners. J Parasitol 73: 811-816.
Araujo A 2000. Paleoparasitology of Chagas disease revaled
Nikolic A, Djurkovic-Djakovic O, Bobic B 1998. Intestinal
by infected tissues from Chilean mummies. Acta Trop 75:
parasitic infections in Serbia. Srp Arh Celok Lek 126: 1-5.
79-84.
OLorcain P, Holland CV 2000. The public health importance
Gade DW 2000. The Cambridge World History of Food. In KF
of Ascaris lumbricoides. Parasitology 121 (Suppl.): S51-
Kiple & KC Ornelas, Cambridge University Press, UK.
71.
Galvin TJ 1968. Development of human and pig Ascaris in the
Pbo S 1985. Molecular cloning of ancient Egyptian mummy
pig and rabbit. J Parasitol 54: 1085-1091.
DNA. Nature 314: 644-645.
Geus F 1995. Archaeology and History of Sai Island. The Sudan
Paggi L, Nascetti G, Orecchia P, Mattiucci S, Bullini L 1985.
Archaeological Research Society Newsletter 8: 27-34.
Biochemical taxonomy of ascaridoid nematodes.
Greenberg JH, Turner II CG, Zegura SL 1986. The settlement
Parassitologia 27: 105-112.
of the Americas: a comparison of the linguistic, dental, and
genetic evidence. Current Anthropol 27: 477-497. Patrucco R, Tello R, Bonavia D 1983. Parasitological studies of
Guhl F, Jaramillo C, Vallejo GA, Yockteng R, Cardenas-Arroyo coprolites of pre-Hispanic Peruvian populations. Curr
F, Fornaciari G, Arriaza B, Aufderheide AC 1999. Isolation Anthrop 24: 393-394.
of Trypanosoma cruzi DNA in 4,000-year-old mummified Rausch R, Tiner JD 1948. Studies on the parasitic helminths of
human tissue from northern Chile. Am J Phys Anthropol the north central states. American Midland Naturalis 39:
108: 401-407. 728-747.
Hagelberg E, Sykes B, Hedges R 1989. Ancient bone DNA Reinhard KJ, Hevly RH, Anderson GA 1987. Helminth re-
amplified. Nature 342: 485. mains from prehistoric Indian coprolites on the Colorado
He L, Min XT, Liu GZ, Xu PB, Li WS 1986. Preliminary Plateau. J Parasitol 73: 630-639.
karyotype studies on Ascaris lumbricoides and Ascaris suum Ruffer MA 1910. Note on the presence of Bilharzia haematobia
from Guangzhou [in Chinese]. Chi Sheng Chung Hsueh Yu in Egyptian mummies of the Twentieth Dynasty (1250-
Chi Sheng Chung Ping Tsa Chih 4: 206-208. 1000 BC). Br Med J 1: 16.
46 Ascariasis in Humans and Pigs O Loreille, F Bouchet

Sauer CO 1952. Agricultural Origins and Dispersals, Ameri- Takata I 1951. Experimental infection of man with Ascaris of
can Geographic Society, New York. man and the pig. Kitasato Arch Exp Med 23: 49-59.
Sprent JFA 1952. Anatomical distinction between human and Tomaso H, Dierich MP, Allerberger F 2001. Helminthic infes-
pig strains of Ascaris. Nature 170: 627-628. tations in the Tyrol, Austria. Clin Microbiol Infect 7: 639-
Stuart MD, Greenspan LL, Glander KE, Clarke MR 1990. A 641.
coprological survey of parasites of wild mantled howling Valentine CC, Hoffner RJ, Henderson SO 2001. Three common
monkeys, Alouatta palliata palliata. J Wildl Dis 26: 547- presentations of ascariasis infection in an urban Emergency
549. Department. J Emerg Med 20: 135-139.