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Malaria infectionand human evolutionL’infezione malarica nel contesto dell’evoluzioneantropologica

Sergio Sabbatani1, Roberto Manfredi1, Sirio Fiorino2

1Infectious Diseases, S. Orsola-Malpighi Hospital, Bologna, Italy;2Internal Medicine, Budrio General Hospital, Budrio, Italy

n INTRODUCTION

The available paleopathological evidencesdemonstrate that infectious diseases as a w-hole became significant as to morbidity and

mortality rates for the human kind, only after thecultural progress which led to the developmentof zootechny, and subsequently to the extensionof agricultural techniques [1, 2]1. The developmentof agriculture and animal breeding characterizedby the domesticating of bovine and cattle and theextensive culture of the most relevant vegetablespecies, led to a progressive process of sedentaryattitude of population2. The demographic devel-opment which followed the increased food avail-ability allowed the first population aggregationsin well circumscribed areas; just in this era, someinfectious diseases (predominantly representedby anthropozoonoses mainly transmitted bybovines, ovines, cattle, and dogs domesticatedduring the Mesolitic age) became epidemic in na-ture, and produced a significant, negative demo-

graphic impact on the resident population. In thisframework, the studies conducted by Cohen andArmelegos demonstrated a strong link betweeninfectious diseases and demographic structure ofpopulations [4].The history of primitive agricultural-based so-cieties, and basically those of the medieval Eu-rope, clearly demonstrate the existing relation-ship with the cyclic temporal sequence of se-vere famine episodes, due to nourishment cri-sis, related to the exaggerated expansion ofpopulations, as opposed to the objective foodavailability, and with a significant relation withdisease epidemics [5]. According to Biraben re-ports, the cycling occurrence of both faminesand epidemics is common of all locations andall ages, since it is supported by some structur-al frailties of the ancient agricultural produc-tion, which were poorly controllable due totheir primitive and backward features [6]3.Should all statements reported above be the re-sult of the process of human development dur-

Leinfezioninella sto-ria dellamedicina

Infectionsin thehistory ofmedicine

Le Infezioni in Medicina, n. 1, 56-74, 2010

1The agricultural development, dated between years 9,000 and 8,000 b.C. in the Middle East allowed to the human kind to rely on a surplus offood-stuff, and therefore the major determinant which limited the demographic growth in the communities which applied to hunting and veg-etable harvesting was not reduced. Otherwise, specialized jobs like the metal manufacturing (which plays a fundamental role in the produc-tion of weapons), became prominent. Subsequently, the history showed the emerging of an aristrocracy devoted to military practices, paral-leling an intellettuale class which addressed its efforts towards the performance of religious-sciamanic rituals In appropriate geographical andenvironmental contexts, the human communities has the opportunity to address their main activities towards merchant arts, and subsequent-ly evolved their commercial trade by sailing boarding the Mediterranean coasts. The Phoenicians represented the earlier, great sailors of theentire Mediterranean sea, and had the merit to develop a first alphabet already during the Bronze age. Approximately in the same time, Ba-bilonian inhabitants discovered the sexagesimal calculation system. 2Demographical estimates, carried out between sixties and seventies of the past century, may lead to hypothesize that European population ac-counted of around eighty million inhabitants, during the mean Neolithic age.3Luigi Capasso, in order to explain the relationship between famines and epidemic illnesses, has developed a theoretical model, based on thecalculation of energy features. The Author underlines that it is indifferent to establish whether the introduction of agriculture was the techno-logical response to a demographic increase, already registered at the end of Paleolithic age, or whether this increase was caused by the earlieranimal and vegetable domestication and culture [3]. According to this model, when the energy amount drawn out of the system (which maybe of low entity in the event of an economy based on hunting and harvesting, or medium entity when the system is founded on agricultureand breeding), becomes lower, compared with the amount of energy needed to nourish and maintain alive the entire population. In this lastevent, a crisis characterize by an extraordinarily elevated mortality rate occurs (it is also the case of epidemics). Based on this theoretical ap-proach, characterized by the binomial demography versus energy taken from the system (i.e. the so-called biomass), we may also fit many his-torical war conflicts, i.e. the barbarian invasions of the Roman empire, or the origin of the World War II by the nazist Germany, especially whenconsidering the invasion of the Soviet Union occurred in the year 1941, attributable to the richness of that country in agricultural and naturalresources (oil- and gas-fields).

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ing the last 10,000-12,000 years, then the rela-tionship between the mankind (Homo habilis,Homo erectus, and Homo sapiens) and differentpathogenic and non-pathogenic microorgan-isms happened since thousands of years. Actu-ally, our ancient progenitors and our ancestorsinteracted in many different, potential wayswith these microorganisms, resulting in differ-ent situations. Generally, we can say that all liv-ing species have reciprocally conditioned them-selves during their respective evolutionaryprocess; as a consequence, following the con-siderations of K.C. Kim, the comprehensivespecies evolution is a “coevolution” process [7].Of course, the reciprocal influences have beengreater when they regarded organisms morestrictly connected to each other, from a tropismpoint of view [3].In order to underline this last concept we canremind some extensively acquired knowledge. The start of a whatsoever reciprocal influence ismatched with a “contact” between two livingbeings belonging to different species, if fromthis contact an interaction occurs, or the two liv-ing beings are not indifferent; in this last event,three different scenarios may be expected, andall of them are characterized by the start of anhistorical “coevolutive” course. In the first case,an organism feeds of the second one (or viceversa), while in the second case both living be-ings live together without any sort of damagefor both organisms; finally, in the third case anorganism makes use of some function or re-source of another living being, for its own ad-vantage. In this third situation, two differentoptions may result: a damage for the second or-ganism which suffers from the presence of thefirst one, may occur or not. The first reported case represents the main as-pect of an elementary physiological function asthe nutrition is; the coevolution mechanismstarts when the “predator” organism developsspecific mechanisms able to support the effica-cy of its aggressive behavior, while in the sametime the “prey” mounts a response, by amelio-rating its specific defence mechanisms (i.e. nat-ural immunity defence).The second event is characterized by the so-called mutualistic symbiosis. This last ancestraloccurrence has its origin in the procariote or-ganisms symbiosis, when these microorgan-isms began to perform differentiated and wellspecialized functions in a common cytoplasmicenvironment.The third case is representative of the parasitic

condition. The parasite organism (smaller whencompared with its host), has a significant ad-vantage by feeding at host’s expenses, and inthis circumstance, should a damage of the hostfollow, an infectious disease occurs, whileshould the parasited organisms have no or neg-ligible damages, it becomes a sort of “healthycarrier” of its specific parasite; notably, this lastcondition may move towards a frank disease ifa state of deterioration and/or immunosup-pression occurs, to support this pathologicalevolution.When coming back to the concept of coevolu-tion, the parasitism represents its ideal model.In fact, in this situation the host-parasite dy-namics finds its major expression, and eachadaptive change of the parasite organism has arespective host reaction. This extremely dy-namic relation, characterized by an action-reac-tion paradigm, imposes evolutive changes to allliving species interested by this process, and isdirectly responsible for the eterochronic varia-tions typical of ontogenesis [3].The host who suffers from a “non-compensable”damage caused by the parasite-infectious agent,are destined to succumb; on the other hand,eventual parasite organisms which cause too rel-evant damage to their specific host have nospace in the evolutionary selection of species.The “ideal” relationship occurs when the varia-tions of one partner represent a selective pres-sure which induce the emerging of an host pop-ulation which is progressively less reactive, anda parallel population of progressively less viru-lent and aggressive parasite organisms.When the parasites are species-specific, an epi-demiological balance becomes evident betweenhost and parasite (evolving toward a parasitismwithout damage/disease, or a symbiotic rela-tionship)4. However, it is also possible that thevirulence pathogenicity features of single para-sites, i.e. their capacity to induce damage tospecific hosts, may be increased by repeatedpassages of the same organisms in host belong-ing to different species. All parasites whichhave a proportionally broader host-range spec-trum are favoured in eluding the selectivemechanisms of epidemiological balance, andmay still cause severe disease forms, despite

4From a theoretical point of view, the older is the first contact withinfectious agents, the milder is expected to be the “modern” disease,since in these last conditions a very long time is elapsed, to allow thedevelopment of a balance between the host and its potential para-site.

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their “old” parasite-host relationship. From thispoint of view, the Influenza virus model is veryrelevant and up to date.As early as in the year 1963, Cockburn summa-rized these concepts as follows: “The infectiousdiseases affecting modern human kind are theresult of all the relationship that our specieshad in the past and still has, with organismswhich acted and still act as parasites” [8]. As awhole, the biological history of human infec-tious diseases had its development thanks totwo different types of relationship: in the firstcase, a dual relationship between man and par-asite (including parasites specific for humans),leads to a parallel, true coevolution of both“partners”. Otherwise, the relationship is ex-tended to hosts other than humans; this last is-sue predominates when the relevant parasitehas a spontaneous, extensive host range [3].These two different modes of relationship wereaffected by temporal changes, so that the hu-man-parasite dynamics has driven in a signifi-cant way the evolution of mankind. On thesepreliminary basis, we can hypothesize that infec-tious diseases have represented a very relevantway of selective pressure in the different envi-ronmental ecosystems, on a worldwide basis,and may have conditioned the ontogeneticmechanisms too, possibly modifying the evolu-tive potential of single species and organisms [3].Through an accurate revision of the existing lit-erature evidences, we will make an attempt toverify whether presently available data mayadd significantly to establish what was (andwhat is) the biological role played by a diffuseinfectious disease like malaria, which repre-sents one of the most relevant infectious disor-ders registered during the long-term evolutionof the genus Homo, considering the species Ho-mo habilis, Homo erectus, and Homo sapiens.

n THE INFECTIONS BY MALARIA PLASMODIA IN NATURE

Malaria plasmodia descend from ancient endo-cellular parasites of the enteric tract of a com-

mon ancestor of reptiles, amphibians, andbirds, all of them parasited by some form ofPlasmodium spp. These endocellular parasitestake origin from ancestral, free forms, which oc-casionally remained trapped by phagocytes ofpluricellular organisms, where they were ableto survive5. Subsequently, the ancestors of plas-modia acquired the ability to transfer from in-testinal cells towards liver cells (through theportal circulation). When colonizing the livertissue, plasmodia might complete their vital cy-cles, and may spread into the systemic circula-tion of their host. The parasite cycle was re-acti-vated when an hematophagous arthropod in-sect6, by stinging the relevant host, became ableto transfer the infection to another individual,which therefore became parasited. On theground of paleontological evidences, we cansuppose that plasmodia, already present since along time before the appearance of the first ver-tebrate beings, circulated among primates,transmitted by mosquitoes of the genus Culex.In the New World, this situation remained un-modified for a long time, during the mean andlate Tertiary ages, until the genus Anopheles ap-peared in the Oligocene period (but only in theOld World), and became the vector of plas-modia infection among Primates. This eventhas been dated at the end of late Tertiary age.Later, during the late Pleistocene age, humansfinally appeared in the African continent, andimmediately became involved by epidemiolog-ical chains of the different plasmodial infec-tions. As a consequence, this assumption ex-plains that the mosquitoes of the genus Culexremained the selected vectors for birds andmammals since the Cenozoic era, while pri-mates and subsequently early hominids weresimply involved in pre-existing epidemiologi-cal chains [1, 3].The comparative genotypic analysis of plas-modia, and in particular the comparison ofDNA contained in the 18S ribosomial gene,suggests that the divergence of Plasmodium fal-ciparum from its more close plasmodial species,the Plasmodium reichenowi which parasiteschimpanzees, occurred approximately five to

5As known, the ability to survive for a variable time into phagocyte cells of the immune system has been demonstrated for different bacterialspecies (i.e. meningococci, gonococci, mycobacteria) [8].6Fossilized mosquitoes have been detected sono in the Eocenic limestone layers of the Green River Formation, in Colorado, USA [9]; in the ma-jority of cases, the insects were represented by the Culex species [10], responsible for the transmission of infection to birds, while, as alreadyknown, the tranmssion of plasmodia among primates is due to the mosquito belonging to the genus Anopheles. Fossilized remnants of this lastgenus were retrieved in the Old World only. Luigi Capasso supposes that the genus Culex, which appeared during early Tertiary age, guar-anteed the spread of pasmodia in this particular time period. Only later, during mean Tertiary age, the circulation of plasmodia will be ensuredby mosquitoes of the genus Anopheles [3].

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ten million years ago, and in this last case a co-evolution has occurred [11]. As opposed to theevolution of Plasmodium falciparum into thethree other species which cause disease in hu-mans (Plasmodium. vivax, Plasmodium malariae,and Plasmodium ovale), the passage of these par-asite could be happened through a collateralpathway, and possibly directly from other pri-mates to humans (Figure 1). An intense debate emerged with regard to themost recent molecular evolution of Plasmodiumfalciparum. A controversial statement points outthat the Plasmodium falciparum populations pre-sent in the third millennium might descendfrom a common ancestor, a still survivingspecies alive a few thousands of years ago dur-ing the Neolitic period. Otherwise, different in-vestigations underline that Plasmodium falci-parum maintained a dimension of a real popu-lation, also incorporating a high degree of ge-netic diversity, during hundred thousand yearsof evolution. These apparently divergent state-ments may be the result of the fact that re-searches based their studies on different data-

bases, and non-coincident methods were ap-plied for molecular biology investigations;moreover, problems linked to a different datingaffected these last examinations. In a recent andmore extensive report, a synthesis of bothpoints of view has been performed, and the Au-thors concluded that the more recent, commonancestor of Plasmodium falciparummay be datedat around one hundred thousand years ago;furthermore, a major expansion of this last plas-modial species should be occurred during thelast 10,000 years [12].On the ground of molecular biology data, thisinfection is recognized as very old in nature. Asmentioned by Mario Coluzzi, its evolution intoa human parasite may be strictly linked to theprogression of its more effective vectors in trop-ical regions of the African continent, such as theexpansion of the mosquitoes group belongingto the species called Anopheles gambiae. Since theecotype living in the forest has been establishedas the oldest one, we may therefore suggest thatan anthropophilic behaviour probably began ina limited forest environment, where the mos-

Figure 1 - A graphic representation of the biological and phylogenetical evolution of Plasmodium vivax, Pla-smodium malariae, and Plasmodium ovale (group a parasites), and the more recent pathway performed byPlasmodium falciparum (group b). The first three mentioned plasmodia completed their coevolution in pa-rallel with that of the mankind or, alternatively, human encountered these protozoa during the most ancientphases of evolution (the so-called structural parasites). On the other hand, Plasmodium falciparum has beenmet during proportionally more recent times, and it has been acquired after spread among monkeys. The Fi-gure is modified from the cited paper by L. Capasso [15].

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quitoes has a small number of great-size ani-mals at disposal, and may have a greater ten-dency to affect humans [13]. The early ho-minids were thought to live in an environmentcharacterized by rain forests as opposed toopen, grassy grounds. Presently, these humanancestors are thought to be already present inWestern and Eastern Africa around six millionyears ago [14]7. The results of these multidisci-plinary researches lead to conclude that the firstcontact between Anopheles gambiae and Homosapiens occurred in the forest ecotype of tropicalAfrica, and this event was not necessarily so re-cent (i.e. dating at the Holocene period), butmay be much older, and may have involvedearlier human ancestors.If biomolecular studies of DNA of Plasmodiumfalciparum suggest that this last parasite is mucholder, the role played by Anopheles gambiae re-mains essential in order to establish how andwhen the malaria spread had its diffusion, sincethe increased growth of Plasmodium falciparumin the population of the Holocene era was prob-ably supported by the evolution of other eco-types of Anopheles gambiae, different from theoriginal ecotype which had its habitat in therain forest [11]. To conclude, the common ancestor which hadits development around one hundred thousandyears ago, is probably related to the demo-graphic expansion of the first modern humans.However, the role of the vector Anopheles gam-biae was determinant in order to make its diffu-sion epidemic, and conditioning by this waythe development of humanity itself. The Figure2 depicts a sample of mosquito, included andpreserved into an amber fragment.On the other hand, Luigi Capasso attributes asignificant role to the mosquito species Culextanzanianae. This last anthropophagite insectmay have been a relevant vector of plasmodiafor our ancestors, since it had its endemic areaalong the East African coast, between the latePliocene and the early Pleistocene, concurrentlywith the presence of the first members of thegenus Homo [5]. An interesting, but still open question, regardswhich species of mosquito played a key role inthe transmission of malaria plasmodia in the

New World, before the exploration carried outby Christopher Columbus. On the ground ofthe above-mentioned dissertation, we may hy-pothesize a separated evolution of mosquitoespresent in the Old World and in the New Worldrespectively, since its parallels the separatedevolution of primates in the two different geo-graphical contexts. A “remixing” of the differ-ent species of both mosquitoes and plasmodiaoccurred in a late period, i.e. when the Euro-Asiatic and the American continents becamecloser, thanks to the practicability of the Beringstrait, and the consequent transfer of humans tothe American continent8; consequently, the ear-lier, separated development of both vectors andplasmodia had the first opportunity to blend,when the New World became finally inhabited.As known, plasmodia extensively affect pri-mates, and a number of simian species suffer-ing from malaria has been identified: the clini-cal picture is characterized by fever andsplenomegaly, but an asymptomatic course isnot infrequent. For instance, the African chim-panzee may be infected by Plasmodium.malariae,and this infection may be subsequently trans-ferred to humans, while American primates areparasited by Plasmodium brasilianum, and alsothis species may infect humans causing a febrileillness similar to quartan fever [15]. Humanmalaria infection is usually well tolerated byprimates (in particular, gorillas and chim-panzees), and also Plasmodium falciparum ofgreater monkeys induces a more benign dis-ease, compared with human one. These differ-ences also indicate that this last Plasmodiumspecies had contacts with non-human primatessince a very long time, and this “old” contactled to a partial resistance to this specific infec-tion.During the evolutionary course, the problem ofthe epidemiological balance between Plasmodi-

Figure 2 - A mosquito specimen included in an am-ber fragment.

7A recent archaeological research points out that human populationsof hunters-harvesters were continuatively present in the environ-ment of rain forest, for a long period of time [14].8The humans are known to haver reached the Americas through theBering strait around 30,000 years ago; however, adequate findingsable to document their presence, are dated at 15,000 years ago.

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um falciparum and human kind has resolved(from an evolutionistic point of view), after thedevelopment of a genetic resistance based onthe establishment and the persistence of a poly-morphism of genes which encode for the globinprotein9.The peculiar features of malaria caused by Plas-modium falciparum in humans, which are exten-sively different from those observed in malariadisease induced by Plasmodium vivax, Plasmodi-um ovale, and Plasmodium malariae, may suggestthat the most extensive contact with Plasmodiumfalciparum occurred in a proportionally more re-cent time, compared with other malaria forms10.It’s a general opinion that the massive humanmalaria infection from animal reservoirs, oc-curred in Africa after the introduction of agri-culture, and the progressive diffusion of asedentary lifestyle [17]. Probably the massivedeforestation carried out in Eastern Africa toacquire cultivable soil, greatly supported themosquito proliferation. Moreover, the great so-matic plasticity of mosquitoes allowed to thesehaematophagous insects to rapidly adapt to thechanging environmental conditions, by occupy-ing the novel ecologic niche ensured by stag-nant waters which progressively took spacearound primitive villages [3, 18].The subsequent step was the modified habits ofsome mosquito species, which ceased their nu-trition of primate blood, evolving towards thenutrition of human blood. This last occurrencewas the crucial point for the disappearance ofPlasmodium falciparum as a major parasite ofwild primates11, to become one of the most im-portant element of natural selection of humankind [3].In our previous discussion, we examined thepossibility that the New World have been inter-ested by Plasmodium falciparum malaria only af-ter the discovery of Columbus occurred in theyear 1492, since possible, prior contacts regardslow populated regions of North America, were

probably sporadic in frequency, and probablynot sufficient to establish a well recognizeddanger of malaria spread. However, an unan-swered question has been posed, of relevantscientific interest: on which basis may be statedthat malaria was a disease unequivocally limit-ed to the Old World, until the year 1492? In or-der to try an answer to this intriguing question,both paleopathology and population geneticsgive us some relevant evidences. As known,malaria (especially in its chronic-relapsingform) induces some characteristic skeleton stig-mas, sometimes absent and/or aspecific, butwhen characteristically recognized, may lead toconfirm its role in these skeleton deformities.These bone sequelae of malaria, named “porot-ic hyperostosis” from their morphologic fea-tures, are common to other conditions of chron-ic anaemia, due to vitamin and/or iron defi-ciencies. Congenital anemias (i.e. thalassemia,sickle cell anaemia, constitutional anaemia)may also lead to a picture of “porotic hyperos-tosis”. Furthermore, also extensive and pro-longed intestinal parasite diseases may inducea malabsorption syndrome, which in its moresevere forms may support pathological dam-ages not different from those of “porotic hyper-ostosis” encountered in human malaria [3].However, even when taking into account theselimitations, the paleopathologic investigationon human remnants may address (togetherwith other proofs) to establish the eventual re-sponsibility of Plasmodium falciparum in sup-porting this pathological condition. Given thatthe genetic-based hemoglobinopathies repre-sent a reliable marker of population involve-ment, should Plasmodium falciparum be endemicsince long time, among American Indian popu-lations12 these alterations were detected onlywhen genetic exchanges with subjects ofAfrican or European origin occurred, whereasthey were absent when these population inter-actions were not recorded in the past [3]. The

9Both sickle-cell anaemia and thalassaemia, with the respective genetic-encoded haemoglobin variants, are the cause of the extremely un-favourable phenotypic conditions, with respect to the survival of plasmodia inside the red blood cells of heterozygous subjects. The selectivepressure exerted by the endemicity of malaria in some regions of sub-Saharan Africa, is responsible for a proportionally elevated incidence ofheterozygote individuals, which bear a “sickle-cell trait” in around 40% of the general population [16].10When considering these three different malaria subtypes, a real epidemiological balance is highly suspected, since a “parallel coevolution”occurred, while in the case of Plasmodium falciparum the result depended on a forced, elevated selective pressure (a situation which does notoccur in great African primates, which usually show a very mild disease course).11The primates, after the introduction of agriculture and the consequent increase of human aggregation, ceased to represent the main and pri-mordial reservoir of malaria, since human are infected only sporadically with simian plasmodia (i.e. Plasmodium brasilianum, Plasmodium simi-um, Plasmodium cynomolgi) [3].12In the Americas, the “porotic hyperostosis”, which has been found in an elevated number of pre-Colombian human remnants, is probably re-lated anemias due to vitamin or iron deficiency, which were frequent after the extension of agriculture, and the diffusion of a corn-based di-etary habit [19, 20]. Other possible causes may be searched among the frequent chronic elminthiases of the gut, and the endemic presence ofbartonellosis in Southern America [22].

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extraction of DNA sequences of Plasmodium fal-ciparum from bone specimens of human skele-tons - which, due to a concurrent “porotic hy-perostosis”, may have been affected by malaria- could contribute to solve this persisting con-troversial issue. Starting from the last decade of the twentiethcentury, the molecular biology techniques al-lowed to reliably establish the existence of anancient Plasmodium falciparum infection. Thelaboratory assay named “ParafsightTM-F”,which is able to retrieve the antigen of the his-tidin-rich protein 2 (the so-called proteinPfHRP-2) descending from the Plasmodium falci-parum trophozoites, may help in the identifica-tion of malaria antigen specific of Plasmodiumfalciparum. In fact, tissue samples collected fromnaturally dried human mummies coming fromEgypt and Nubia (dating around 5,200 and1,450 years ago), were tested positive at thePfHRP-2 protein search, while tissue samplescoming from subjects detected in the “Ca-marones” site, located in the Atacama desert(South America-Chile), and dating around theyear 1,000 b.C., were proved negative at thesame laboratory search [22].As we may synthetically assess, although bio-molecular investigation have reached very ad-vanced scientific standards, the assessment re-garding the ancient history of malaria infectionis still subject to some uncertainty. In order toreach our objectives, it becomes easier to traceback the ancient figures starting from the pre-sent knowledge, linking the scientific evidencescoming from molecular biology, with bothavailable historical-medical evidences, and ar-chaeological materials and related studies (in-cluding paleopathological human remnants, orarchaeological findings found in sites exploredduring proportionally recent years).When limiting our considerations to the Euro-pean continent, we may reasonably supposethat during the glacial periods of the Quater-nary era (around two million years ago) untilthe Mesolitic era, malaria remained absent fromour continent, since it is a temperature-depen-dent disease, and mosquitoes were not able tosurvive in this climatic environment. Subse-quently, the quotations regarding intermittentfevers reported by the ancient literature (Hip-pocratical reports), dating at fourth and fifthcentury b.C., some old Indian documents (ofdifficult dating), and the Chinese literature(dating around the first millenium b.C.), do notleave doubts that the benign tertian malaria

(due to Plasmodium vivax) and the quartan fever(caused by Plasmodium malariae), were alreadyendemic in Greece, India, and China. We maytherefore reasonably think that these benignmalaria forms reached Greece between the endof the last glaciations era, and the year 500 b.C.,although no certain documents exist [11].While there is a general agreement in retainingthe origin of the malignant tertian malaria (dueto Plasmodium falciparum) located in tropicalAfrica [23], the Roman historical Celsus (wholived in the first half of the first century a.C.)[24], made the first differential diagnosis be-tween the clinical course of Plasmodium falci-parum-related fever, and the more benign Plas-modium vivax-related disease. The awarenessthat already during the Roman age a substan-tial difference between the two main forms ofmalaria fever had been recognized, comes fromthe documents of the ancient physician Celsus,as reported around the year 100 a.C. by Archi-gene of Apamea13. Of certain, the physicians ofthe ancient Roman world were already awarethat patients suffering from a malignant semi-tertian fever had a worse course in autumn, andthis complication was not so frequent in olderages. This consideration suggests that in Italythe pathocoenosis included a novel disease,clearly identified for the first time in this histor-ical period, and the different clinical variants ofmalaria were endemic in central and southernItaly [11].Mirko Grmek, one of the most important con-temporary experts in the history of Medicine,on the ground of literature evidences, suggeststhat just malaria fevers interested the Athenianarmy which besieged the Sicily city of Syracuse,during the Peloponnese war [26]. On theground of this observation taken from the liter-ature, and re-enforced by the knowledge of al-ready existing marshes around the city of Syra-cuse, we can reasonably state that 2,500 yearsago malaria was already endemic in Sicily. Nu-merous colonies founded by Greek populationsin Southern Italy between year 800 and year 600b.C., during subsequent decades were severelyaffected by malaria epidemics: in particular, thehistorical city of Paestum, where extraordinarytemples and monuments were built betweensixth and fifth century b.C., experienced a rapiddecline just because of malaria epidemics [11].After an earlier historical phase, which in-volved the centuries preceding 400 b.C. (limited

13Quoted from Aetius of Amida (former Mesopotamiam region) [25].

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to Sicily and Southern Italy in its coast sites),the malaria diffused in Italy in a South-Northdirection toward central-western regions, whenthe so-called second stage of malaria spreadhad its origin [11]. During the initial time peri-od, until around 400 b.C., the Etruscan cities ofMaremma region in Italy, had their period ofexpansion, as demonstrated by the relevant ar-chaeological findings like vases imported byGreece. Moreover, continued and close contactswith the emerging Roman potency character-ized this period (in fact, the last three Romankings were all of Etruscan origin). In these loca-tions, it is possible that malaria spread alongTuscany coasts during the third and secondcentury. Cato the Senior refers on the presenceof malaria fever in Graviscae, the port of theEtruscan city of Tarquinia, around the year 150b.C. [27]. Probably plasmodia and their vectorsreached the northern Latium coasts (central-western Italy), facilitated by sea transfers fromSardinia, Sicily, or directly from NorthernAfrica. We remind that during the precedingcenturies the citizens of Syracuse and theGreeks, after defeating the Etruscans in thenaval battle of Cuma (474 b.C.), achieved thecontrol of the Thyrrenian sea, exerting theircheck on maritime routes and commercial ex-changes with regions where malaria was al-ready an endemic disease, since long time. Dur-ing late second century b.C. in the city of Rome,Asclepiade of Bitinia described severe, inter-mittent febrile illnesses, sometimes complicatedby central nervous system involvement, andGalen quotes this information in its writings,three centuries after [28]. Rome was certainlysurrounded by territories infested by malariaparasites also during the imperial centuries,and when considering also the general declinefollowing the dissolution of Roman Empire,even more during subsequent centuries.Archive studies of the Vatican correspondence,demonstrated that pilgrims coming fromNorthern Europe around the eight century a.C.,preferred to make their way to Rome duringthe winter season, in order to avoid “the badair” which polluted the environment during thesummer period of the year [29]. The way fol-lowed by malaria parasites towards North-Eastern Italy lasted for a while. Around 2,000years ago the region of the Po river delta, notfar from the city of Ravenna (the last capital ofthe Western Roman Empire), was reported tobe free from malaria. This last informationcomes from the writers Vitruvio and Strabone,

and sounded strange for the time, since the sur-rounding regions were already marshy [30, 31].According to Sidonio Apollinare, should mos-quitos be present, they were probably not suffi-ciently effective in transmitting malaria para-sites: in fact, malaria became endemic in theseregion only during the Middle Ages, when ananthropophilic mosquito (Anopheles sacharovi),appeared and became permanent along theNorth-Eastern Italian coasts [32]. In these re-gions, the malaria epidemic had a very slowspread, and according to Sallares and cowork-ers, it happened in a range of 1,500 years, fromyear 500 b.C. and year 1,000 b.C. [11]. Sinceboth Plasmodium vivax and Plasmodium malariaewere present in the Mediterranean Europe be-fore the appearance of Plasmodium falciparum, aselection and an extensive spread of an effectivevector was needed to support their stable circu-lation. The speciation of a mosquito belongingto the Anopheles gambiae complex made proba-bly possible the step toward an endemic behav-iour of malaria [33]. In fact, only when an effi-cient vector, capable to survive to cold temper-atures of North-Eastern Italy, was selected,Plasmodium falciparum and the two other Plas-modium species had the possibility to take rootin geographic areas which were morefavourable to their features (often followingfield abandonment typical of the Middle Agesperiod14.

n DIRECT BIOMOLECULAR EVIDENCES OF THE PRESENCE OF MALARIA IN THE ANCIENT WORLD

In 2008 a study reporting very reliable data ofidentification of the ancient malaria DNA (aD-NA) has been published, together with the evi-dence of Plasmodium falciparum in Egyptianmummies dated back to 4,000 years ago [34].The Authors analyzed 91 bone tissue samplesof a parallel number of mummies and skeletonsretrieved in the different Egyptian sites15.

14In other terms, as Sallares and coworkers wrote, “…all the threespecies of Plasmodium were ready to move together, simultaneouslytoward novel regions, but they had to wait for appropriate vectormosquitoes in order to start moving” [11].15The most ancient site is the pre- and the proto-dynastical site ofAbido (an High Egyptian town) (dating around years 3,500-2,800 b.C.), the second site is represented by a grave in West Thebes, of theMean Kingdom time (years 2,050-1,650 b. C.), while the third siterefers to different tomb complexes, also localized at West Thebes,which have been built between the mean and novel Kingdom, anduntil later periods (from 2,050 up to 500 b. C.).

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All specimens have been tested for Plasmodiumspp. DNA with a polymerase chain reaction(PCR) technique which is based on the use ofrDNA of 18S ribosomal subunits, in order toidentify unambiguously the eventual presenceof an ancient malaria infection. Moreover, intwo human remnants coming from two differ-ent sites of the New Kingdom, a positivity formalaria infection was found, with specimensmatching with osteopathological signs ofchronic anaemia [34]. The same assay per-formed on tissue samples obtained from rem-nants belonging to earlier time periods, provednegative for malaria infection [34]. On the ground of obtained results, the Authorscritically notice that the studies performed dur-ing recent years probably overestimated the realamount of malaria infection in the ancientEgyptian world [22]. These studies employedthe so-called antigen-2 (a protein rich of his-tidin), which was detected in over 40% of testedspecimens, and in 92% of specimens of humanremnants, where signs of the “porotic hyperos-tosis” were appreciable [22]. Even taking into ac-count this criticism, the same Authors conclud-ed that Plasmodium falciparum infection played asignificant role in conditioning the limited lifeexpectancy of the ancient Egyptian populations,on the basis of molecular biology examinationsconducted with PCR techniques [34].As mentioned in the above paragraph, if exist-ing evidences document the slow, progressivespread of malaria from Southern to North-East-ern Italy, multiple scientific evidences cumulat-ed during the last years confirm this last theory.Between years 1988 and 1992, an archaeologicalsite has been discovered in Lugnano in Teveri-na, a small town located in the Tiber river val-ley16, located 70 miles far from Rome. This siteincludes an extraordinarily large children

cemetery of the Roman Italy17, dating at around450 b.C. [35, 36]. Archeological evidencebrought the Authors to conclude that deathswere secondary to an epidemic disease whichacted in a proportionally short time period,probably during the Summer18 (the season bur-dened by the highest risk of malaria diffusion),as established on the ground of the paleobio-logic analysis of remnants of plants and seedsretrieved in the graves [27].Of 47 human remnants retrieved, 22 were rep-resented by premature children19, and also themajority of other dead bodies were fromneonates. As anticipated, both environmentalfeatures and the characteristics of the retrieval,induced the researchers to establish that neona-tal deaths were due to a severe malaria. Thisstatement has been examined by experts likeMario Coluzzi, who confirmed this opinion, onthe basis of environmental, biological, and en-tomological considerations [11]. Molecular biol-ogy assays were also performed, through theextraction of DNA genome of Plasmodium falci-parum from bone specimens. In one case (i.e.burying number 36) the extraction of plasmodi-al ribosomal DNA was attained by amplifyingtwo different specimens. This last case regardsa female child who was aged 2-3 years, at thetime of death [39]. The feasibility of DNA am-plification of Plasmodium falciparum from 1,500-year-old skeleton, suggests a massive malariainfection20, and not an asymptomatic-pau-cisymptomatic course due to a low parasiteload. This last result of relevant scientific valueled to conclude that this death was caused bymalaria, thus demonstrating that the Tiber riv-er valley was a place of an epidemic malariaaround the year 450 b.C. [11, 39]. The Authorsfinally observe that the presence of Plasmodiumfalciparum in central Italy, probably associated

16The archaeological site is located at 185 meters above the sea in a hill area, 3.5 km far from the river [35]. The river valleys represented theearliest malaric areas in Italy, at the end of Western Roman empire, since the river beds were not maintained, and when overflows occurred,very favourable sites for mosquito replication appeared, especially when flow waters retired leaving small or large ponds. In the specific caseof Lugnano, this small town is located a few kilometres far from the immission of the Orte river into the Tiber river, so that this particular ge-ographical area was at greater risk for malaria spread.17In these archaeological cemeteries, findings referring to spells were found, like the presence of skeletons of dog puppies, which maybe servedas a weapon against devils, which were thought to be responsible for children’s lethal disease. The term “abracadabra”, of common use in mag-ic rituals, were primarily employed as a spell against tertian fever, as reported by the writer Quinto Sereno [37]. We also remind that childreninfrequently received a burial during Roman time. It makes these retrievals of extremely relevant interest, since they allow to advance pale-opathological, anthropological, and archaeological questions.18Some accurate liteature evidences exist regarding the reknown unhealthyness of these areas. During the Summer of the year 467 SidonioApollinare run along Italy from Ravenna to Rome, to encounter the emperor Antemio. During his journey, he just reached the malaric regionsof ancient Umbria and Tuscany, leaving a striking evidence of the infamous effects of “poisonous miasmas”, severe fever, and insatiable thirst,all clear witnesses of malaria disease.19Malaria predisposes not only to elevated mortality rates, but also a striking predisposition to absorption among pregnant women, causing anintrauterine growth retardation. Until now, absorption is found among both immune and partially immune pregnant women, in holoendem-ic tropical regions of the world.20According to the Authors, the low rate of positive retrievals, may be attribited to the poor preservation of plasmodial DNA in ancient bonespecimens [11].

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with its main vector, i.e. Anopheles labranchiae(still present in the Tiber river valley during theWorld War II), significantly increased the riskof developing malaria, of the resident popula-tion [27]. Since these Italians regions were al-ready populated, epidemic events of malariawere expected at that time.

n INDIRECT GENETIC EVIDENCES OF MALARIA SPREAD IN THE ANCIENTWORLD

Genetic population studies performed duringthe past 25 years gave us the elements to un-derstand which was the era of initial selectivepressure operated by malaria on Mediterraneanpopulations. The most recent data confirmedthe previous suspicions, i.e. this pressure start-ed in proportionally recent times. This observa-tion is acceptable since during previous timesthe climate was too cold (at least in Europe, andduring glacial ages), to allow malaria spread. When underlying that the most ancient osteolo-gy evidence of thalassemia, characterized by thetypical “porotic hyperostosis”, has been first de-tected in a site close to the village of Atlit Yam,quite far from the Israel coastal line, and datingaround 10,000 years ago, population genetics al-lowed to find a lack of aplotype diversity whichis usually associated with the largest number ofmutations conferring resistance to malaria in themodern Mediterranean populations [40]. Theseevidence suggest that these changes probablyrepresented an evolution started from a time notbeyond 4,200 years ago [41]. All major determi-nants of inherited genetic resistance were al-ready present in the Mediterranean basin at thetime of Roman empire. In particular, studiescarried out in Italy on a paediatric skeleton, pre-sumably dating between fifth and seventh cen-tury b.C., and those performed on human rem-nants of a 17-year-old woman dating at the third

century b.C., discovered in Pisa, and in a Romanvilla located in the town of Settefinestre (Tus-cany coast), showed bone abnormities typical ofthalassemia [42, 43]. The same elements havebeen noticed in 11 human skeletons exhumed inthe Pantanello necropolis, close to the Greekcolony of Metaponto [44]: all of them were af-fected by the typical signs of “porotic hyperos-tosis”, dating between the fourth and the fifthcentury b.C., and were probably homozygotesfor thalassemia21. This temporal gap in the re-trieval of the same somatic stigmata in humanremnants underlines that the Greek colonizers22

were the first population inhabitating Italy, topresent a significant frequency of thalassemia-related genetic mutations, and the consequentbone marrow involvement, as well as a relation-ship with anaemia caused by Plasmodium falci-parum disease.To confirm the early presence of the typical, in-direct somatic signs of endemic malaria inNorthern Africa and in the Middle East, is ofgreat help the retrieval of a skeleton of a 20-year-old male with “porotic hyperostosis”, lo-cated in the island of Failaka, in the PersianGulf. The bones, which have been dated by ra-diocarbon techniques at approximately2,130±80 years ago, allowed to find fossilizederythrocytes with the typical sickle shape, withthe aid of electron microscopy studies [46]. Weremind that the haemoglobin S is still presentwith a 2% frequency in some Mediterraneanpopulations, and the aplotype analysis demon-strated that the sickle cell trait is present in Sici-ly, Northern Greece, and Western Arabia, andare reversely linked with the aplotype called“Benin” (detected in Central-Western Africa[47]. According to these genetic populationstudies, the remnants retrieved in the Failaka is-land may demonstrate that these findings couldbelong to a descendant of a Macedonian soldiercoming from Northern Greece, and carrying thecharacteristic aplotype Benin23 [38].

21The children who were homozygote for thalassemia, i.e. those who inherited the trait from both parents, suffer from the clinical variant calledthalassemia major, or Cooley’s anemia. They show a voluminous skull with swelling in the frontal and parietal regions, a large forehead, pre-minent zygomas, distanced eyes often with an almond-shape aspect, a large and flabby nose, and protruding jaws. This aspect is reproducedon some terracotta heads, like those retrieved in Smyrna archaeological remnants, close to the Anatolian cost of Egean sea (Figure 3).22Observations carried out on archaeological specimens carefully assessed from an historical medicine point of view, already confirmed thatGreeks frequently showed the typical somatic tracts of thalassemia. Mirko Grmek and Danielle Gourevitch examined seven small terracottastatues discovered again in Smyrna, on the Anatolian coast of Turkey, dating around the Hellenistic period. All these manufacts preserved“swelling faces, with hypertrophic zygomas, and symmetric prominences on selected (frontal-parietal) parts of the head, and also had an en-larged, flattened nose basis” (Figure 3). Based on these somatic aspects, the two researchers concluded that these small statues presented thepatognomonic traits of homozygote thalassemia [45].23Alternatively to this last hypothesis, it remains possible that the remnants of the indigenous human retrieved in Failaka was a thalassemiacarrier who, because of the molecular features of the sickle cell trait (with a different aplotype association), had a genotypic mutation origi-nating from India and Arabia [48]. We remind that the Indian variant of thalassemia has a phenotype associated with elevated plasma levelsof fetal hemoglobin, which inhibits the formation of sickle erythrocytes. On the ground of this second hypothesis, we may explain why the sub-ject retrieved in the Persian Gulf area, could have reached the adult age.

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The deficit of glucose-6-phosphate dehydroge-nase (G6PD) enzyme ranks third in importance,among genetic human mutations which arecommonly encountered in the Mediterraneanpopulations, and confer intrinsic resistanceagainst Plasmodium falciparum infection. Theabove-mentioned children of Lugnano havebeen selected for the presence of the two mostcommon G6DP mutations encountered inMediterranean populations. The results of thesedemanding studies of population genetic(which are not extensively reported), led to the

conclusion that G6PD deficiency, together withthe thalassemia trait and the sickle cell anemiatrait, were already present in Southern Europeat the decline of Roman empire. It probably oc-curred as an evolutionistic response of popula-tions to the pressure exerted by the malaria en-demic [11].The route of malaria endemic may be also in-vestigated through the detection of mutationswhich were particularly frequent, and are typi-cal of thalassemia among present Mediter-ranean populations, like the B+IVSnt110 (G-A)mutation, which is also found in some in Italianregions colonized by ancient Greeks. This mu-tations has its major and greater frequency inthe countries which border the EasternMediterranean basin like Greece, while inTurkey an elevated levels of aplotype diversitywas also demonstrated [41].On the ground of this genetic drift, some Au-thors suggested that this particular mutationhad its origin in Greece or Anatolia, and subse-quently moved towards Western countries, in-cluding Italy, following the migration routesoriginating from the Hellenic peninsula startingfrom the eighth century b.C. [49].On the contrary, the CD39 mutation, which rep-resents the second most frequent mutation en-countered in Mediterranean populations bear-ing the beta-thalassemia trait, is concentrated inthe Western Mediterranean regions colonizedby Phoenicians and achieves its higher level ofvariety through genetic recombinations associ-ated with the aplotype [49]. On the ground ofthese prevalence, we now think that this muta-tion had its roots in Northern Africa24, and laterspread towards the major Italian islands andthe Iberian peninsula, at the time of Phoeniciancolonization [11]. Therefore, population genetic studies support-ed the scientific evidence of two very relevantsubjects. Malaria, its mosquito vectors25, and

Figure 3 - A model of burying in the paleochristiancemetery of Lugnano (Tiber river valley). The child’sskeleton is contained into an amphora. The image isreproduced from the paper of L. Sallares et al. [11].

Figure 4 - Child’s head bearing the typical stigmas ofCooley’s anaemia. This archaeological report hasbeen retrieved in Smyrna, Aegean coast of Turkey(Paris, Louvre Museum).

24During the glacial era, Northern Africa presumably representedthe main shelter of Anopheles lambrachiae, and still today it remainsthe geographic area which allows the preservation of the most af-fordable habitat for these varieties. We also remind that Anopheleslambrachiae is known to be the most efficient vector of malaria in ourcountry (Italy) during recent centuries.25The most important malaria vectors in Western and WasternMediterranean basin were represented by two different mosquitospecies: Anopheles labranchiae and Anopheles sacharovi. The firstspecies is the most efficient vector in Central and Southern Italy, itoriginated in Northern Africa, and although remaining active allyear round it is reproduced in this geographic area only. The secondspecies, called Anopheles sacharovi, comes from near Orient, and wasthe predominant vector in Greece; it succeeds in surviving the coldwinter temperatures of some regions of Southern Europe, being ca-pable of hibernation [50].

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human genetic mechanisms conferring resis-tance to malaria infection, reached SouthernEurope through two different routes. The firstone took origin from Tunisia, and goingthrough Sardinia and Sicily arrived in SouthernItaly, and subsequently moved along the Italianpeninsula along a Southern-Northern route.The second route started from Eastern Mediter-ranean coast, moved towards West from Ana-tolia to Greece, and finally reached Italy26 [11]. The second subject, which supports the title it-self, we have selected for our present contribu-tion, leads to understand that Plasmodium falci-parum infection certainly represented an impor-tant agent of natural selection, by acting initial-ly on populations living in the Eastern Mediter-ranean coasts, while this pressure moved to-wards Italy only in a second time. During thisprogression of malaria towards Italian peninsu-la, an important role was played, as mentioned,by the two distinct varieties of Anopheles mos-quitoes which represented the major vectors ofplasmodia in Southern and Central Italy(Anopheles sacharovi), and Northern-EasternItaly (Anopheles labranchiae), respectively. Thelater appearance of malaria in North-WesternItaly compared to Greece, is related to the slowadaptive mechanisms of Anopheles labranchiae,which spent some centuries to match climateconditions present in regions where an efficientvector was still lacking, although the majorityof conditions which support the spread of werealready present. Given these premises, the pro-gressive diffusion of malaria in continental Italyoccurred during the late Middle Ages, whenthe endemic became evident in the Po riverdelta, and the North-Eastern regions of Italy27.

The close relationship of Ravenna (and laterVenice), with the Byzantine empire allowed theenvironmental penetration of Anophelessacharovi mosquito, while Anopheles labranchiae,which was the predominating vector of West-ern countries (including Central and SouthernItaly), did not succeed in overcoming the barri-er represented by the Tuscany-Emilian Apen-nines [11].During the late Middle Ages, central Italy washeavily interested by the malaria endemic.Dante Alighieri himself, in his Divine Comedy(twenty-ninth chapter of the “Inferno” “(Hell)section of the poem), clearly reports the healththe poor healthy conditions of people living inthe Chiana river valley, Maremma, and Sar-dinia between the months of July and Septem-ber, in the poem triplets referred to the “Male-bolge” (“Malebolge”) location28. The Figure 5represents Dante and Virgil on a high steepplace, while they look and pity the damnedsouls of false manufacturers, who weredamned to spend their eternal punishment inthis Hell round of the Divine Comedy.According to research studies completed untilnow, the times and modes of malaria spread inItaly appear reasonably known, while it is notthe case for the malaria diffusion in Greece andEastern Mediterranean countries. Based on lit-erature sources, the text of the well known“Corpo Ippocratico” (Hippocratic corpus) ofthe fifth century b.C. are the most ancient andreliable reference, which state the involvementof Greece in the malaria epidemic29. On the oth-er hand, until now we still lack of certain bio-molecular evidences which may confirm itspresence in older times30.

26According to the historical literature which could not exploit genetic population studies, older scientist suggested that malaria reached Italyand Greece during the same time period, since it was not possible to imagine that this epidemic infection actually followed different routes anddifferent historical evolutions in two countries which are very close and apparently so similar from a climatic-environmental point of view [11].27Already after the flood of 489 a.C., a small group of Benedectine monks settles in the island of Codigoro located in th former Po river delta,to witness their faith through their known motto “ora et labora” (pray and work). Later in the history, during the year 874, these monk com-munity was steadily located at the renown Pomposa abbey, which has been founded by these Benedectine monastery (as recorded in a lettersent from the Pope John VIII to the Emperor). Thanks to the work of Benedectine monks, the Po valleys were periodically reclaimed from stag-nant water (marshes). During the centuries, the advocacy of Benedectine and Cistercian monks was that to impede the degradation of lands inItaly and in many European countries, therefore preventing the massive spread of malaria, although a huge tribute was paid in terms of hu-man life, by monks themselves.28“Quando noi fummo sor l’ultima chiostra / di Malebolge, sì che i suoi conversi / potean parere a la veduta nostra, / lamenti saettaron me diversi, / che pietàferrati avean li strali; / ond’io li orecchi con le man copersi / Qual dolor fora, se de lì spedali / di Valdichiana tra ‘l luglio e ‘l settembre / e di Maremma e diSardigna i mali / fossero in una fossa tutti ‘nsembre, / tal era quivi, e tal puzzo n’usciva / qual suol venir de le marcite membre”. (When we stood above the final cloister / of Malebolge / and all of its lay brothers became discernible to us, / strange arrows of lament, theirshafts, / with pity at their tips, pierced me, / so that I pressed my hands against my ears. / If the contagion of every hospital / in Valdichiana,from July until September, / and in the Maremma and Sardinia, / were amassedin one malarial ditch, / such suffering was in that place. /And from it rose the stench of festering limbs).29Mirko Grmec and Danielle Gourevitch gave their interpretation of paintings which depict Heracles when he kills the mythological Hidrasnake with on some Corinthians ceramics, dating from years 630 to 570 b.C. (Figure 5), as an attempt to reclaim the malaria-infested marchesof Lema country, in Argus region [45].30Although molecular biology studies able to date the involvement of Greece in the malaria epidemic are not available, the observations re-garding the retrieval of the typical “porotic hyperostosis” remain the most significant reference to re-build the prehistory of malaria in the Hel-lenic peninsula [11, 51, 52].

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Indirectly, we can infer that Greece was in-volved by malaria spread, from the study ofsome archaeological findings retrieved in the“Magna Grecia” colonies, where the early ar-rival of Greek colonists determined their mag-nificent flourishing31; as a consequence, we cansupposed that the Homer’s native country wasalready involved by the worse effects of malar-ia endemic.

In Central-Southern Italy, the malaria-inducedpressure was certainly precocious, and a factorwhich greatly contributed to a favourable envi-ronmental context was represented by a tem-perature increase, occurring concurrently withthe iron age and remained until the eight cen-tury b.C., and leading to a raising of the marinelevel of approximately one meter [52]. Such arapid elevation of marine surface supported theformation of coastal marshes along theMediterranean basin, in particular when asandy soil was of concern (i.e. close to the rivermouths). An interest occurrence involved theItalian Campania region, and in particular theSele river valley, where Etruscans built theirSouthern outpost (located in the town of Pon-tecagnano). The Sele river, together with itstributaries, was subject to frequent overflow-ing, and these events probably acted in favouror the intensification of malaria spread in thisterritory. The archaeological excavation of animportant Etruscan necropolis, including twodistinct groups of buried bodies, the first of 275subjects who spent their life between the sev-enth and the sixth century b.C., and the secondaccounting of 398 dead bodies, dating betweenthe fifth and the fourth century b.C., allowedboth paleopathological and paleonutritional in-vestigations, which demonstrated frequentmalnutrition syndromes, related to a predomi-nant anaemia picture, where the typical form of“porotic hyperostosis” was proved proportion-ally frequent [57]. These paleopathological evi-dences were detected in 8.72% of subjects in thefirst mentioned group, and in 3.72% of deadbodies belonging to the second group [58]. Theconcomitant diffusion of thalassemia in thispopulation was thought to be proportionallyfrequent, although the reclamations and the as-sessment of water and soils realized in theselands by Etruscans32 enabled an amelioration of

Figure 5 - Geographic chart of Italy, published in theyear 1882 with fittings by Luigi Torelli. The spreadroutes of malaria are represented, as well as theareas primarily involved in the malaria epidemics.

31While the relationship between Southern Italy and Greece are certain, one more suggestion regards the possible, common origin of Etruscansand some Anatolic populations (coming from Lydia), coming from Egean sites, based on archeological studies and recent genetic populationinvestigations. With regard to archaeological documentation, it is mainly represented by the Lemnos stele, located in the so named island ofthe northern Egean sea, where it was retrieved in the year 1885. This stele reports a text including 198 digits, which give origin to 33 to 40words, and whose epicoric idiom is strictly related with the Etruscan language. Furthermore, excavations carried out in the year 1928 by theItalian school of Athens lead to the discovery of vasa fragments of local production which bear similar writings. These archeological pieces re-markably demonstrate that such a language was actually written and spoken in the Lemnos island, thus leading to strong arguments in favorof the Oriental origin of the Etruscan people, as concurrently stated by the writer Tucidide, and other evidences [53]. Italian genetists analyzedthe genotypic features of subjects coming from Tuscany, in particular the towns of Murlo and Volterra, and the Casentino valley, and com-pared them with those of human samples coming from Sicily, Sardinia, Southern Balkans, and Anatolia. The results of these investigationsdemonstrated that the specimens coming from Murlo and Volterra are more strictly related to those of the populations of near Eastern coun-tries, compared with those coming from other Italian regions. In particular, a genetic variant close to that retrieved in Anatolian residents on-ly has been found in Murlo inhabitants, and from a general point of view the specimens from Tuscany show a close affinity with those of theLemnos island [54, 55]. Data coming from population genetic studies confirm the writings of the ancient Herodotus, i.e. that Etruscans hadtheir roots in the ancient Lydia [56].32The Etruscans managed skilled engineering techniques [59]. The Roman historical writer Titus Livy (liber XXVII, 46, 5) reports on a “Greekditch” (digged channels), aimed to comney waters from the lower course of Clanio river, until the open sea.

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environmental healthy, and consequently of the“porotic hyperostosis”. Even though these re-searchers did not allow to establish with ab-solute certainty the presence of an endemicmalaria in this geographic area, the proportion-ally elevated frequency of the “porotic hyperos-tosis” found in dead bodies underlines the sus-picion that a significant percentage of deathswas due to malaria infection, in a high-risk en-vironment.

n SOME PATTERNS OF CULTURE FITTINGTO ENDEMIC MALARIA IN HUMAN POPULATIONS

Although the main adaptive measures of popu-lations against malaria infection were mainly ofgenetic and biological origin, however the con-tributions provide by cultural fitting was veryrelevant and interesting to be investigated. Thislast adaptation involved also an extremely rele-vant aspect of daily human life, such as the di-etary habits. In the light of knowledge coming from nutri-tional sciences, the alimentary choices operatedin the traditional diet by populations who livein endemic malaria regions, during time wereprogressively addressed towards food whichallow a greater protection33 against plasmodiaparasites. Certainly, a dietary regimen enrichedby broad bens (Vicia faba), typical of countriessurrounding the Mediterranean sea, confers aremarkable increase of defences against malar-ia infection [61, 62]. In fact, some broad bensmetabolites, released from the gastrointestinaltract, act as potent oxidant agents, which mayinduce relevant in vitro antimalaric effects, witha further increase of potency when G6PD defi-ciency is concurrently present [63]. An experi-mental case-control study conducted in Thai-land demonstrated that the dietary intake ofbroad bens significantly increases antimalaricdefence, especially in subjects who are homozy-gotes or heterozygotes for the synthesis of thehaemoglobin E variant [64].Also vitamin C (ascorbic acid), may act as a pro-oxidizing compound, in presence of an amount

of active oxide-reducing iron, so that this lastcompound has a well recognized anti-malariceffect, exerted by acting on the advanced devel-opment stages of plasmodia [60]. Although acontrolled clinical trial failed in demonstratinga clinically recognized antimalaric activity ofdiet supplemented with notable amounts of vi-tamin C [65], there is no doubt that Mediter-ranean diet usually rich of ascorbic acid-con-taining food, may be responsible for some pro-tection against malaria, and this cultural basis

Figure 6 - Dante and Virgilio (from the Dante Ali-ghieri “Divine Comedy”) are mercifully looking todamned souls confined in the tenth Hell’s “bolgia”,and damnes because of they were “false manufac-turers of every piece of work”.

Figure 7 - A representation of an Hydria retrieved inCaere (close to the Central Italian town of Cervete-ri), and dating at year 525 b.C. The mythological kil-ling of Lerna’s Hydria is represented (preserved atPaul Getty Museum of Malibu (CA, USA).

33Without entering the complicated biochemical mechanisms which act on the plasmodia parasite versus erythrocite relationship, three maincomponents played a significant role in the adaptation of dietary profile which involved the subjects living in endemic areas: i) the dietary in-take of “oxidative nourishment” profile, which is the primary mediator of an anti-malaria diet based on oxidative principles; ii) the presenceof adequate or elevated dietary levels of transitional metals bearing an active oxidizing-reductive potential (i.e. iron and copper), where ironwas particularly useful to catalyse the production of free radicals starting from “oxidative food”, through the so-called Haber-Weiss reaction;iii) the selection of a dietary pattern which minimized the intake of vitamins and other compounds which play an ant-oxidant activity in theparasited red blood cells [60].

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contributed to reduce the disease risk, as exten-sively reported by the literature [60, 66, 67].An elevated number of contribution, whoseconclusions are herewith briefly summarized,point out that a low dietary intake of iron repre-sent a sort of adaptive measure of human popu-lation against malaria, and other infectious dis-eases, too. In fact, the iron is an essential elementenabling the replication of plasmodia, and hu-mans may lower plasma iron levels by increas-ing its binding with a series of plasma proteins,including lattoferrin, transferrin, and ferritine,which are particularly concentrated in the milk.Starting from the 1970s, some studies seemed tosuggest that subjects suffering from a chroniciron dietary deficiency had a lower incidence ofmalaria, and also a lower parasitic load, in theevent of malaria infection. Since milk is a rele-vant dietary component in populations histori-cally engaged in animal breeding, and milk con-tains an elevated amount of riboflavin34, also di-etary regimens based on milk and its derivativeswas thought to represent a preventive measureagainst malaria parasites [60].Furthermore, a diet based on an increased in-take of fish (in particular, sardines andpilchard, and related species), which is rich inhighly pro-oxidative fats, like the omega-3eicosapentanoid acid, and the docosaexanoicacid, when associated with the intake of foodwith a proportionally low vitamin K content,has been demonstrated capable to induce a spe-cific protective function against malaria. TheMediterranean populations of historical agescould certainly take advantage from an elevat-ed dietary intake of fish, especially in coastalzones. Also the frequent resort to salt downand/or kipper the available fishes (like cod andherring), increased their availability also duringthe Winter season, when fishing became dan-gerous and poorly profitable.The preservation of currently employed food(i.e. that of grain and derivatives, in mud con-tainers), allowed to mount exogenous defencesagainst malaria in a population living in Nubiabetween the years 350 and 550 b.C. Certainly,

the Ballam population was not aware of suchan advantage. In fact, agriculture products likewheat, barley, and millet, which were the basisfor the production of bear and bread, after theirpreservation in vases shaped with Nubianmud, were colonized by streptomycetes, lead-ing to a massive growth of these bacteria. Strep-tomycetes35 are known to produce natural an-tibiotics like streptomycines [68], which act ef-fectively against Gram-negative and Gram-pos-itive bacteria, Rickettsiae, and Spirochetae, aswell as malaria plasmodia, too [69]. Althoughthe Nubian, Ballam population could not beaware of these notices, the consumption of foodcontaminated with the above-mentioned mi-croorganisms allowed to introduce undoubtfulselective advantages in the prevention ofmalaria which was a major paediatric morbidi-ty and mortality factor, in this geographicalcontext [68]. The observation of nature repre-sented one of the fundamental elements of an-cient populations, with particular reference toEgyptians. This last population, thanks to theirlarge cultural and archeological state, have leftextensive documentation, which reached thecontemporary world. Even when consideringmalaria, Egyptians were the first ancient popu-lation which has transmitted a written witnessof their knowledge. Into the Denderah36 temple,hieroglyphic characters compose the followingsentence: “Beware of going outside home afterthe sunset, during the weeks following Nilerive floods”. This last sentence certainly repre-sents the oldest anti-malaric preventive pre-cept, and it demonstrates that this evolved peo-ple already had developed their nature obser-vation, and already retrieved relevant precepts,aimed to ameliorate their quality of life.Also literature evidences allow us to appreciatethe depth of ancient Egyptian knowledge. TheGreek writer Erodoto (II, 95) reports that Egyp-tians, who had an intensive fishing practice,were able to defend themselves from mosqui-toes. He reports: “Against mosquitoes, whichare extremely numerous, they adopted thesecontrivances: people inhabiting over a marsh are

34Although riboflavin is usually considered as an antioxydant, it was actually though to act as a non-specific pro-oxidant. Some in vitro evi-dences show a riboflavin oxydant activity against malaria, which appeared similar to that exerted by artemisin, when using a special culturemedium which includes eryhtrocites stratified according to age, and alpha- and beta-thalassemic traits [60].35Streptomycetes require a very dry, warm, and alkaline environment to support their growth. They include 60-70% of bacteria retrieved in thedesert soil of Nubia. These microorganisms live by metabolising the organic particles of the soil, and have a positive advantage as opposed toother bacterial species, which have a more rapid growth, but are strictly dependent on a wet and acid environment, and deserve moderate tem-peratures.36The rear portion of the Denderah temple has been built as early as at the end of the second century b.C., during the kingdom of Ptolemy thetwelfth Auletes, from year 80 to 51 b.C.: he was the father of Cleopatra the seventh, the last queen of the Ptolemaic dynasty. During the Ro-man age some other buildings were added, at the time of Augustus, Nero, Domitian, and Trajan emperors.

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protected by high towers, where they go andsleep overnight; in fact, because of wind mos-quitoes cannot fly towards to height. Citizensliving in the proximity of marshes have foundexpedients other than towers: everybody has gota net, which is used for fishing during the daytime; overnight, the same net is placed aroundthe bed where they sleep under an effective pro-tection. Although we know that mosquitoesmay prick also through a coat or a sheet, theseinsect cannot even attempt to go through a net”.In conclusion, we can state that the adaptivemechanisms of the human kind never stoppedduring the time, but the evolution of a series ofbiological factors (plasmodia, vectors), paral-leled the environmental changes, by searchingconditions which were more favourable to their“success”. On the other hand, formerly ho-minids, and later humans, through a series ofgenetic and immunological adaptations (whichhave not been extensively treated in the presentreview), and other cultural and behavioural fit-tings, guaranteed an adequate evolutionary re-sponse, able to successfully overcome the pres-sure imposed by the malaria parasite dissemi-nation. However, it remains possible that just at

this time a Plasmodium species which usuallyinfects primates, possibly through a novel, ef-fective vector, may become able to infect hu-mans, either in a rain forest, or an isolated barepatch, or around a tropical pond located in cen-tral Africa, Southern America, or Borneo37. Inthese hypothetical circumstances, one or moreselected mutations, capable to confer to thisnovel plasmodia both human tropism and anincreased pathogenicity, may occur. Theseevent may open a new pathway to a novel epi-demic, caused by the first introduction into avillage or a community of a novel Plasmodiumspecies, which may act dramatically on thehealth of the susceptible mankind (which lacksof a specific immune system defence), after be-ing steadily adapted to the local environment.

Key words: malaria, plasmodia parasites, an-thropology, evolution, human kind.

AcknowledgementsThe Authors wish to thank Dr. Simonetta Righi (In-ternal Medicine Section, Centralized Medical Li-brary, University of Bologna, Bologna, Italy), for herexpert advice and valuable co-operation.

37In the year 2004 a first focus of malaria due to Plasmodium knowlesi has been recorded in the Sarawak state of Indonesia, and in the Borneo re-gion of Malaysia, and between November 2004 and Marchg 2005 four death were described in the Sarawak area, initially attributed to a Plas-modium malariae infection. Some doubts raised regarding this attributed aetiology, since Plasmodium malariae usually is associated with a lowparasitemia and it does not lead to death. The blood samples of these patients were retrospectively examined by a PCR technique, and the ex-istence of this novel human Plasmodium species was confirmed. Noticeably, Plasmodium knowlesi shares a similar microscopic aspect just withPlasmodium malariae, and it is therefore at risk to be missed or underestimated in its frequency, and especially in its pathogenicity [70]. In a veryrecent paper published in the outstanding Journal “Nature”, the genotypic sequence of Plasmodium knowlesi has been reported for the first time[71]. At the same time, N.J. White wrote an “Editorial comment” for the Journal of the Infectious Disease Society of America, named “ClinicalInfectious Diseases”, which was entitled: “Plasmodium knowlesi: The Fifth Human Malaria Parasite” [72].

During the evolution of the genus Homo, with re-gard to the species habilis, erectus and sapiens, malar-ia has played a key biological role in influencing hu-man development. The plasmodia causing malariahave evolved in two ways, in biological and phylo-genetic terms: Plasmodium vivax, Plasmodium malari-ae and Plasmodium ovale appear to have either coe-volved with human mankind, or encountered hu-man species during the most ancient phases of Ho-mo evolution; on the other hand, Plasmodium falci-parum has been transmitted to humans by monkeysin a more recent period, probably between the endof the Mesolithic and the beginning of the Neolithicage. The authors show both direct and indirect bio-molecular evidence of malarial infection, detectedin buried subjects, dating to ancient times andbrought to light in the course of archaeological ex-cavations in major Mediterranean sites. In this review of the literature the authors present

scientific evidence confirming the role of malariain affecting the evolution of populations inMediterranean countries. The people living in sev-eral different Mediterranean regions, the cradle ofwestern civilization, have been progressively in-fluenced by malaria in the course of the spread ofthis endemic disease in recent millennia. In addi-tion, populations affected by endemic malaria pro-gressively developed cultural, dietary and behav-ioural adaptation mechanisms, which contributedto diminish the risk of disease. These habits wereprobably not fully conscious. Nevertheless it maybe thought that both these customs and biologicalmodifications, caused by malarial plasmodia,favoured the emergence of groups of people withgreater resistance to malaria. All these factors havediminished the unfavourable demographic impactof the disease, also positively influencing the gen-eral development and growth of civilization.

SUMMARY

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Lungo il corso dell’evoluzione del genere Homo (speciehabilis, erectus e sapiens), la malaria ha giocato un ruo-lo biologico chiave sullo sviluppo antropologico. I Pla-smodi malarici a loro volta si distinguono per due per-corsi evolutivi, da un punto di vista biologico e filoge-netico. In particolare, Plasmodium vivax, Plasmodiummalariae, e Plasmodium ovale, si sarebbero caratterizza-ti per una co-evoluzione con il genere umano, o avreb-bero parassitato la specie umana nelle fasi più antichedell’evoluzione del genere Homo. D’altra parte, Plasmo-dium falciparum è stato trasmesso all’uomo dai primatiin periodi storici più recenti, probabilmente tra la finedell’era Mesolitica e l’inizio dell’era Neolitica. Gli Au-tori riportano evidenze biomolecolari dirette ed indiret-te dell’esistenza dell’infezione malarica, rilevata in se-polture del Mondo Antico, portate alla luce nel corso discavi archeologici effettuati in importanti siti del bacinodel Mediterraneo. In questa rassegna della letteratura,vengono organizzate le evidenze di letteratura attual-mente disponibili: esse confermano il ruolo svolto dallamalaria nell’influenzare l’evoluzione delle popolazioni

residenti nel bacino del Mediterraneo. Popolazioni chevivevano in diverse regioni del bacino del Mar Mediter-raneo, che rappresentavano il fulcro della civiltà occi-dentale, hanno subíto una progressiva influenza da par-te dell’infezione malarica, nel corso della diffusione diquesta malattia endemica nei passati millenni. Oltre aciò, popolazioni interessate da forme endemiche di mala-ria, hanno progressivamente sviluppato meccanismi diadattamento di ordine culturale, alimentare, e compor-tamentale, che contribuivano a ridurre il rischio di con-trarre il rischio di tale parassitosi. Questi mutamenti diabitudine non erano probabilmente attuati in piena con-sapevolezza. Ciò nonostante, si può pensare che sia icambiamenti comportamentali sia le modificazioni bio-logiche causate dalla presenza di plasmodi malarici, ab-biano favorito l’emergere di gruppi di popolazione dota-ti di più elevati livelli di resistenza nei confronti dellamalaria stessa. Tutto questo insieme di fattori ha infinecontenuto l’impatto demografico sfavorevole della ma-lattia, influenzando nel contempo in misura positiva ilprogresso e lo sviluppo generale della civiltà.

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