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275 years since the epidemic of plague in Cluj: Dr. Alexandru Lenghel’s contribution to its investigation
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Publication date (Electronic):
15 November 2015
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Clujul Medical
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Abstract
Plague is one of the most impressive diseases in the cultural history of mankind. Its lethality has influenced the evolution of society and it is frequently represented in fine arts and literature.
The principality of Transylvania was also affected by this infection, the plague having strongly impacted both economic and social development.
Between 1738 and 1739 an important plague epidemic spread in Transylvania. The authors introduce and discuss a less known work on this epidemic, with focus on its impact on the city of Cluj - a book written in 1930 by Dr. Alexandru Lenghel, who later became a target of political persecution during the Stalinist period, while his work entered a cone of shadow.
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Lessons from the History of Quarantine, from Plague to Influenza A
Eugenia Tognotti (2013)
The risk for deadly infectious diseases with pandemic potential (e.g., severe acute respiratory syndrome [SARS]) is increasing worldwide, as is the risk for resurgence of long-standing infectious diseases (e.g., tuberculosis) and for acts of biological terrorism. To lessen the risk from these new and resurging threats to public health, authorities are again using quarantine as a strategy for limiting the spread of communicable diseases ( 1 ). The history of quarantine—not in its narrower sense, but in the larger sense of restraining the movement of persons or goods on land or sea because of a contagious disease—has not been given much attention by historians of public health. Yet, a historical perspective of quarantine can contribute to a better understanding of its applications and can help trace the long roots of stigma and prejudice from the time of the Black Death and early outbreaks of cholera to the 1918 influenza pandemic ( 2 ) and to the first influenza pandemic of the twenty-first century, the 2009 influenza A(H1N1)pdm09 outbreak ( 3 ). Quarantine (from the Italian “quaranta,” meaning 40) was adopted as an obligatory means of separating persons, animals, and goods that may have been exposed to a contagious disease. Since the fourteenth century, quarantine has been the cornerstone of a coordinated disease-control strategy, including isolation, sanitary cordons, bills of health issued to ships, fumigation, disinfection, and regulation of groups of persons who were believed to be responsible for spreading the infection ( 4 , 5 ). Plague Organized institutional responses to disease control began during the plague epidemic of 1347–1352 ( 6 ). The plague was initially spread by sailors, rats, and cargo arriving in Sicily from the eastern Mediterranean ( 6 , 7 ); it quickly spread throughout Italy, decimating the populations of powerful city-states like Florence, Venice, and Genoa ( 8 ). The pestilence then moved from ports in Italy to ports in France and Spain ( 9 ). From northeastern Italy, the plague crossed the Alps and affected populations in Austria and central Europe. Toward the end of the fourteenth century, the epidemic had abated but not disappeared; outbreaks of pneumonic and septicemic plague occurred in different cities during the next 350 years ( 8 ). Medicine was impotent against plague ( 8 ); the only way to escape infection was to avoid contact with infected persons and contaminated objects. Thus, some city-states prevented strangers from entering their cities, particularly, merchants ( 10 ) and minority groups, such as Jews and persons with leprosy. A sanitary cordon—not to be broken on pain of death—was imposed by armed guards along transit routes and at access points to cities. Implementation of these measures required rapid, firm action by authorities, including prompt mobilization of repressive police forces. A rigid separation between healthy and infected persons was initially accomplished through the use of makeshift camps ( 10 ). Quarantine was first introduced in 1377 in Dubrovnik on Croatia’s Dalmatian Coast ( 11 ), and the first permanent plague hospital (lazaretto) was opened by the Republic of Venice in 1423 on the small island of Santa Maria di Nazareth. The lazaretto was commonly referred to as Nazarethum or Lazarethum because of the resemblance of the word lazaretto to the biblical name Lazarus ( 12 ). In 1467, Genoa adopted the Venetian system, and in 1476 in Marseille, France, a hospital for persons with leprosy was converted into a lazaretto. Lazarettos were located far enough away from centers of habitation to restrict the spread of disease but close enough to transport the sick. Where possible, lazarettos were located so that a natural barrier, such as the sea or a river, separated them from the city; when natural barriers were not available, separation was achieved by encircling the lazaretto with a moat or ditch. In ports, lazarettos consisted of buildings used to isolate ship passengers and crew who had or were suspected of having plague. Merchandise from ships was unloaded to designated buildings. Procedures for so-called “purgation” of the various products were prescribed minutely; wool, yarn, cloth, leather, wigs, and blankets were considered the products most likely to transmit disease. Treatment of the goods consisted of continuous ventilation; wax and sponge were immersed in running water for 48 hours. It is not known why 40 days was chosen as the length of isolation time needed to avoid contamination, but it may have derived from Hippocrates theories regarding acute illnesses. Another theory is that the number of days was connected to the Pythagorean theory of numbers. The number 4 had particular significance. Forty days was the period of the biblical travail of Jesus in the desert. Forty days were believed to represent the time necessary for dissipating the pestilential miasma from bodies and goods through the system of isolation, fumigation, and disinfection. In the centuries that followed, the system of isolation was improved ( 13 – 15 ). In connection with the Levantine trade, the next step taken to reduce the spread of disease was to establish bills of health that detailed the sanitary status of a ship’s port of origin ( 14 ). After notification of a fresh outbreak of plague along the eastern Mediterranean Sea, port cities to the west were closed to ships arriving from plague-infected areas ( 15 ). The first city to perfect a system of maritime cordons was Venice, which because of its particular geographic configuration and its prominence as a commercial center, was dangerously exposed ( 12 , 15 , 16 ). The arrival of boats suspected of carrying plague was signaled with a flag that would be seen by lookouts on the church tower of San Marco. The captain was taken in a lifeboat to the health magistrate’s office and was kept in an enclosure where he spoke through a window; thus, conversation took place at a safe distance. This precaution was based on a mistaken hypothesis (i.e., that “pestilential air” transmitted all communicable diseases), but the precaution did prevent direct person-to-person transmission through inhalation of contaminated aerosolized droplets. The captain had to show proof of the health of the sailors and passengers and provide information on the origin of merchandise on board. If there was suspicion of disease on the ship, the captain was ordered to proceed to the quarantine station, where passengers and crew were isolated and the vessel was thoroughly fumigated and retained for 40 days ( 13 , 17 ). This system, which was used by Italian cities, was later adopted by other European countries. The first English quarantine regulations, drawn up in 1663, provided for the confinement (in the Thames estuary) of ships with suspected plague-infected passengers or crew. In 1683 in Marseille, new laws required that all persons suspected of having plague be quarantined and disinfected. In ports in North America, quarantine was introduced during the same decade that attempts were being made to control yellow fever, which first appeared in New York and Boston in 1688 and 1691, respectively ( 18 ). In some colonies, the fear of smallpox outbreaks, which coincided with the arrival of ships, induced health authorities to order mandatory home isolation of persons with smallpox ( 19 ), even though another controversial strategy, inoculation, was being used to protect against the disease. In the United States, quarantine legislation, which until 1796 was the responsibility of states, was implemented in port cities threatened by yellow fever from the West Indies ( 18 ). In 1720, quarantine measures were prescribed during an epidemic of plague that broke out in Marseille and ravaged the Mediterranean seaboard of France and caused great apprehension in England. In England, the Quarantine Act of 1710 was renewed in 1721 and 1733 and again in 1743 during the disastrous epidemic at Messina, Sicily ( 19 ). A system of active surveillance was established in the major Levantine cities. The network, formed by consuls of various countries, connected the great Mediterranean ports of western Europe ( 15 ). Cholera By the eighteenth century, the appearance of yellow fever in Mediterranean ports of France, Spain, and Italy forced governments to introduce rules involving the use of quarantine ( 18 ). But in the nineteenth century, another, even more frightening scourge, cholera, was approaching ( 20 ). Cholera emerged during a period of increasing globalization caused by technological changes in transportation, a drastic decrease in travel time by steamships and railways, and a rise in trade. Cholera, the “Asiatic disease,” reached Europe in 1830 and the United States in 1832, terrifying the populations ( 21 – 24 ). Despite progress regarding the cause and transmission of cholera, there was no effective medical response ( 25 ). During the first wave of cholera outbreaks, the strategies adopted by health officials were essentially those that had been used against plague. New lazarettos were planned at western ports, and an extensive structure was established near Bordeaux, France ( 26 ). At European ports, ships were barred entry if they had “unclean licenses” (i.e., ships arriving from regions where cholera was present) ( 27 ). In cities, authorities adopted social interventions and the traditional health tools. For example, travelers who had contact with infected persons or who came from a place where cholera was present were quarantined, and sick persons were forced into lazarettos. In general, local authorities tried to keep marginalized members of the population away from the cities ( 27 ). In 1836 in Naples, health officials hindered the free movement of prostitutes and beggars, who were considered carriers of contagion and, thus, a danger to the healthy urban population ( 27 , 28 ). This response involved powers of intervention unknown during normal times, and the actions generated widespread fear and resentment. In some countries, the suspension of personal liberty provided the opportunity—using special laws—to stop political opposition. However, the cultural and social context differed from that in previous centuries. For example, the increasing use of quarantine and isolation conflicted with the affirmation of citizens’ rights and growing sentiments of personal freedom fostered by the French Revolution of 1789. In England, liberal reformers contested both quarantine and compulsory vaccination against smallpox. Social and political tensions created an explosive mixture, culminating in popular rebellions and uprisings, a phenomenon that affected numerous European countries ( 29 ). In the Italian states, in which revolutionary groups had taken the cause of unification and republicanism ( 27 ), cholera epidemics provided a justification (i.e., the enforcement of sanitary measures) for increasing police power. By the middle of the nineteenth century, an increasing number of scientists and health administrators began to allege the impotence of sanitary cordons and maritime quarantine against cholera. These old measures depended on the idea that contagion was spread through the interpersonal transmission of germs or by contaminated clothing and objects ( 30 ). This theory justified the severity of measures used against cholera; after all, it had worked well against the plague. The length of quarantine (40 days) exceeded the incubation period for the plague bacillus, providing sufficient time for the death of the infected fleas needed to transmit the disease and of the biological agent, Yersinia pestis. However, quarantine was almost irrelevant as a primary method for preventing yellow fever or cholera. A rigid maritime cordon could only be effective in protecting small islands. During the terrifying cholera epidemic of 1835–1836, the island of Sardinia was the only Italian region to escape cholera, thanks to surveillance by armed men who had orders to prevent, by force, any ship that attempted to disembark persons or cargo on the coast ( 27 ). Anticontagionists, who disbelieved the communicability of cholera, contested quarantine and alleged that the practice was a relic of the past, useless, and damaging to commerce. They complained that the free movement of travelers was hindered by sanitary cordons and by controls at border crossings, which included fumigation and disinfection of clothes (Figures 1,2,3). In addition, quarantine inspired a false sense of security, which was dangerous to public health because it diverted persons from taking the correct precautions. International cooperation and coordination was stymied by the lack of agreement regarding the use of quarantine. The discussion among scientists, health administrators, diplomatic bureaucracies, and governments dragged on for decades, as demonstrated in the debates in the International Sanitary Conferences ( 31 ), particularly after the opening, in 1869, of the Suez Canal, which was perceived as a gate for the diseases of the Orient ( 32 ). Despite pervasive doubts regarding the effectiveness of quarantine, local authorities were reluctant to abandon the protection of the traditional strategies that provided an antidote to population panic, which, during a serious epidemic, could produce chaos and disrupt public order ( 33 ). Figure 1 Disinfecting clothing. France–Italy border during the cholera epidemic of 1865–1866. (Photograph in the author's possession). Figure 2 Quarantine. The female dormitory. France–Italy border during the cholera epidemic of 1865–1866. (Photograph in the author's possession). Figure 3 The control of travelers from cholera-affected countries, who were arriving by land at the France–Italy border during the cholera epidemic of 1865–1866. (Photograph in the author's possession). A turning point in the history of quarantine came after the pathogenic agents of the most feared epidemic diseases were identified between the nineteenth and twentieth centuries. International prophylaxis against cholera, plague, and yellow fever began to be considered separately. In light of the newer knowledge, a restructuring of the international regulations was approved in 1903 by the 11th Sanitary Conference, at which the famed convention of 184 articles was signed ( 31 ). Influenza In 1911, the eleventh edition of Encyclopedia Britannica emphasized that “the old sanitary preventive system of detention of ships and men” was “a thing of the past” ( 34 ). At the time, the battle against infectious diseases seemed about to be won, and the old health practices would only be remembered as an archaic scientific fallacy. No one expected that within a few years, nations would again be forced to implement emergency measures in response to a tremendous health challenge, the 1918 influenza pandemic, which struck the world in 3 waves during 1918–1919 (Technical Appendix). At the time, the etiology of the disease was unknown. Most scientists thought that the pathogenic agent was a bacterium, Haemophilus influenzae, identified in 1892 by German bacteriologist Richard Pfeiffer ( 35 ). During 1918–1919, in a world divided by war, the multilateral health surveillance systems, which had been laboriously built during the previous decades in Europe and the United States, were not helpful in controlling the influenza pandemic. The ancestor of the World Health Organization, the Office International d’Hygiène Publique, located in Paris ( 31 ), could not play any role during the outbreak. At the beginning of the pandemic, the medical officers of the army isolated soldiers with signs or symptoms, but the disease, which was extremely contagious, quickly spread, infecting persons in nearly every country. Various responses to the pandemic were tried. Health authorities in major cities of the Western world implemented a range of disease-containment strategies, including the closure of schools, churches, and theaters and the suspension of public gatherings. In Paris, a sporting event, in which 10,000 youths were to participate, was postponed ( 36 ). Yale University canceled all on-campus public meetings, and some churches in Italy suspended confessions and funeral ceremonies. Physicians encouraged the use of measures like respiratory hygiene and social distancing. However, the measures were implemented too late and in an uncoordinated manner, especially in war-torn areas where interventions (e.g., travel restrictions, border controls) were impractical, during a time when the movement of troops was facilitating the spread of the virus. In Italy, which along with Portugal had the highest mortality rate in Europe, schools were closed after the first case of the unusually severe hemorrhagic pneumonia; however, the decision to close schools was not simultaneously accepted by health and scholastic authorities ( 37 ). Decisions made by health authorities often seemed focused more on reassuring the public about efforts being made to stop transmission of the virus rather than on actually stopping transmission of the virus ( 35 ). Measures adopted in many countries disproportionately affected ethnic and marginalized groups. In colonial possessions (e.g., New Caledonia), restrictions on travel affected the local populations ( 3 ). The role that the media would play in influencing public opinion in the future began to take shape. Newspapers took conflicting positions on health measures and contributed to the spread of panic. The largest and most influential newspaper in Italy, Corriere della Sera, was forced by civil authorities to stop reporting the number of deaths (150–180 deaths/day) in Milan because the reports caused great anxiety among the citizenry. In war-torn nations, censorship caused a lack of communication and transparency regarding the decision-making process, leading to confusion and misunderstanding of disease-control measures and devices, such as face masks (ironically named “muzzles” in Italian) ( 35 ). During the second influenza pandemic of the twentieth century, the “Asian flu” pandemic of 1957–1958, some countries implemented measures to control spread of the disease. The illness was generally milder than that caused by the 1918 influenza, and the global situation differed. Understanding of influenza had advanced greatly: the pathogenic agent had been identified in 1933, vaccines for seasonal epidemics were available, and antimicrobial drugs were available to treat complications. In addition, the World Health Organization had implemented a global influenza surveillance network that provided early warning when novel influenza (H2N2) virus, began spreading in China in February 1957 and worldwide later that year. Vaccines had been developed in Western countries but were not yet available when the pandemic began to spread simultaneously with the opening of schools in several countries. Control measures (e.g., closure of asylums and nurseries, bans on public gatherings) varied from country to country but, at best, merely postponed the onset of disease for a few weeks ( 38 ). This scenario was repeated during the influenza A(H3N2) pandemic of 1968–1969, the third and mildest influenza pandemic of the twentieth century. The virus was first detected in Hong Kong in early 1968 and was introduced into the United States in September 1968 by US Marines returning from Vietnam. In the winter of 1968–69, the virus spread around the world; the effect was limited and there were no specific containment measures. A new chapter in the history of quarantine opened in the early twenty-first century as traditional intervention measures were resurrected in response to the global crisis precipitated by the emergence of SARS, an especially challenging threat to public health worldwide. SARS, which originated in Guangdong Province, China, in 2003, spread along air-travel routes and quickly became a global threat because of its rapid transmission and high mortality rate and because protective immunity in the general population, effective antiviral drugs, and vaccines were lacking. However, compared with influenza, SARS had lower infectivity and a longer incubation period, providing time for instituting a series of containment measures that worked well ( 39 ). The strategies varied among the countries hardest hit by SARS (People’s Republic of China and Hong Kong Special Administrative Region; Singapore; and Canada). In Canada, public health authorities asked persons who might have been exposed to SARS to voluntarily quarantine themselves. In China, police cordoned off buildings, organized checkpoints on roads, and even installed Web cameras in private homes. There was stronger control of persons in the lower social strata (village-level governments were empowered to isolate workers from SARS-affected areas). Public health officials in some areas resorted to repressive police measures, using laws with extremely severe punishments (including the death penalty), against those who violated quarantine. As had occurred in the past, the strategies adopted in some countries during this public health emergency contributed to the discrimination and stigmatization of persons and communities and raised protests and complaints against limitations and travel restrictions. Conclusions More than half a millennium since quarantine became the core of a multicomponent strategy for controlling communicable disease outbreaks, traditional public health tools are being adapted to the nature of individual diseases and to the degree of risk for transmission and are being effectively used to contain outbreaks, such as the 2003 SARS outbreak and the 2009 influenza A(H1N1)pdm09 pandemic. The history of quarantine—how it began, how it was used in the past, and how it is used in the modern era—is a fascinating topic in history of sanitation. Over the centuries, from the time of the Black Death to the first pandemics of the twenty-first century, public health control measures have been an essential way to reduce contact between persons sick with a disease and persons susceptible to the disease. In the absence of pharmaceutical interventions, such measures helped contain infection, delay the spread of disease, avert terror and death, and maintain the infrastructure of society. Quarantine and other public health practices are effective and valuable ways to control communicable disease outbreaks and public anxiety, but these strategies have always been much debated, perceived as intrusive, and accompanied in every age and under all political regimes by an undercurrent of suspicion, distrust, and riots. These strategic measures have raised (and continue to raise) a variety of political, economic, social, and ethical issues ( 39 , 40 ). In the face of a dramatic health crisis, individual rights have often been trampled in the name of public good. The use of segregation or isolation to separate persons suspected of being infected has frequently violated the liberty of outwardly healthy persons, most often from lower classes, and ethnic and marginalized minority groups have been stigmatized and have faced discrimination. This feature, almost inherent in quarantine, traces a line of continuity from the time of plague to the 2009 influenza A(H1N1)pdm09 pandemic. The historical perspective helps with understanding the extent to which panic, connected with social stigma and prejudice, frustrated public health efforts to control the spread of disease. During outbreaks of plague and cholera, the fear of discrimination and mandatory quarantine and isolation led the weakest social groups and minorities to escape affected areas and, thus, contribute to spreading the disease farther and faster, as occurred regularly in towns affected by deadly disease outbreaks. But in the globalized world, fear, alarm, and panic, augmented by global media, can spread farther and faster and, thus, play a larger role than in the past. Furthermore, in this setting, entire populations or segments of populations, not just persons or minority groups, are at risk of being stigmatized. In the face of new challenges posed in the twenty-first century by the increasing risk for the emergence and rapid spread of infectious diseases, quarantine and other public health tools remain central to public health preparedness. But these measures, by their nature, require vigilant attention to avoid causing prejudice and intolerance. Public trust must be gained through regular, transparent, and comprehensive communications that balance the risks and benefits of public health interventions. Successful responses to public health emergencies must heed the valuable lessons of the past ( 39 , 40 ). Technical Appendix List of publications chronicling the influenza pandemic of 1918–1919.
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Genotyping, Orientalis-like Yersinia pestis, and Plague Pandemics
Michel Drancourt, Véronique Roux, La Dang … (2004)
Yersinia pestis, a group A bioterrorism agent ( 1 ), causes plague, a reemerging zoonotic disease transmitted to humans through flea bites and typically characterized by the appearance of a tender and swollen lymph node, the bubo ( 2 ). This organism has been subdivided into three biovars on the basis of their abilities to ferment glycerol and to reduce nitrate. Based on their current geographic niche and on historical records that indicate the geographic origin of the pandemics, researchers have postulated that each biovar caused a specific pandemic ( 2 , 3 ). Biovar Antiqua, from East Africa, may have descended from bacteria that caused the first pandemic, whereas Medievalis, from central Asia, may have descended from the bacteria that caused the second pandemic. Bacteria linked to the third pandemic are all of the Orientalis biovar ( 3 ). In this study, we tested this hypothesis for the first time by detecting biovars in ancient human remains. No molecular biology–based method proved reliable and convenient for Y. pestis genotyping. Genome sequences of Y. pestis strain CO92, a Orientalis biovar, and Y. pestis strain KIM, a Medievalis biovar, are now available ( 4 , 5 ), which provides an opportunity to examine them for differences associated with the biovar and for genotyping. Genome analysis of the closely related Rickettsia prowazekii ( 6 ) and R. conorii ( 7 ) showed that intergenic spacers, which have been submitted to less evolutionary pressure than coding sequences, may be variable enough to differentiate closely related microorganisms. We, therefore, hypothesized that sequencing of several intergenic spacers would allow determination of a biovar-specific spacer pattern in Y. pestis. We named this method multiple spacer typing (MST). We first demonstrated that MST allowed biovar genotyping of a large collection of Y. pestis isolates and further applied it to the dental pulp collected from persons whose deaths are attributed to the first and second pandemics. Methods Bacterial Strains Thirty-five strains representative of the three Y. pestis biovars (11 Antiqua isolates, 12 Medievalis isolates, and 12 Orientalis isolates) isolated from 1947 to 1996 from various host species in 13 countries are presented in Table 1. Nineteen of these isolates have been previously characterized by Achtman et al. ( 8 ). Nucleic acid was extracted as previously described ( 9 ), and species identification was confirmed for all the strains by partial sequencing of the rpob gene ( 10 ). Table 1 Alleles of eight spacers in three Yersinia pestis biovars Biovar YP no. strains Country YP1 YP3 YP4 YP5 YP7 YP8 YP9 YP10 Isolate type Antiqua 611/Japan Japan 1 4 1 3 1 1 1 1 1 552/Margaret Kenya 1 3 1 1 4 1 1 1 2 548/343 Belgium 1 3 1 1 5 1 1 1 3 544 Congo 1 3 1 1 7 1 1 1 4 549 Kenya 1 3 1 1 8 1 1 1 5 542 Belgium 1 3 1 1 6 1 1 1 6 550 Congo 1 3 1 1 9 1 1 1 7 553 Kenya 1 3 1 1 7 1 1 1 4 566 Kenya 1 3 1 1 6 1 1 1 6 677 Kenya 1 3 1 1 9 1 1 1 7 545 Kenya 1 3 1 1 7 1 1 1 4 Medievalis 519/PKH-4 Kurdistan 2 2 2 2 1 1 1 1 10 616/PAR-13 Iran 2 2 2 2 1 1 1 1 8 557/PKR292 Kurdistan 2 2 2 2 4 1 1 1 9 564 Kurdistan 2 2 2 2 6 1 1 1 10 565 Turkey 2 2 2 2 5 1 1 1 8 557 Kurdistan 2 2 2 2 4 1 1 1 9 518 Kurdistan 2 2 2 2 5 1 1 1 8 520 Kurdistan 2 2 2 2 5 1 1 1 8 560 Kurdistan 2 2 2 2 5 1 1 1 8 561 Kurdistan 2 2 2 2 5 1 1 1 8 617 Iran 2 2 2 2 5 1 1 1 8 670 Kurdistan 2 2 2 2 5 1 1 1 8 1594 Kurdistan 2 2 2 2 9 1 1 1 11 Orientalis 304/6-69 Madagascar 1 5 1 1 1 2 2 1 12 685 Germany 1 5 1 1 2 2 2 1 13 Hamburg10 USA 1 5 1 1 2 2 2 2 14 CO92 USA 1 5 1 1 2 2 2 1 13 507 Vietnam 1 1 1 1 6 2 2 1 15 1513 Madagascar 1 5 1 1 2 2 2 1 16 571 Brazil 1 5 1 1 4 2 2 1 17 613 Myanmar 1 5 1 1 3 2 2 1 18 643 Madagascar 1 5 1 1 3 2 2 1 18 695 Germany 1 1 1 1 4 2 2 1 17 772 Vietnam 1 5 1 1 4 2 2 1 17 989 Vietnam 1 5 1 1 1 2 2 1 19 Spacer Sequence Database and Phylogenetic Analyses We analyzed the complete genome sequences of Y. pestis strain CO92, biovar Orientalis (GenBank accession no. NC-003143) ( 4 ) and Y. pestis strain KIM, biovar Medievalis (GenBank accession no. NC-004088) ( 5 ), which were obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( 11 ). We used the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) to determine the primer sequences specific for the genomic segments of interest ( 12 ). The primers flanked intergenic sequences of Y. pestis CO92 that exhibited large sequence differences with the homologous Y. pestis KIM strain sequences. We generated a list of Y. pestis CO92 intergenic sequences of 50 to 300 bp and carried out BLASTN searches to identify the homologous intergenic sequences in Y. pestis KIM strain by using the Y. pestis CO92 genes flanking the intergenic sequences as queries ( 13 ). When both genes flanking the intergenic sequence exhibited best-matches with the BLAST score >120 bits, we estimated the length of the corresponding intergenic sequence in the Y. pestis KIM strain. We then aligned the homologous intergenic sequences ( 8 x 106 combinations (223 molecular events). The 387-bp spacer YP1 exhibited a 183-bp deletion specific for the Medievalis isolates. For spacer YP3, we observed five alleles: Orientalis isolates 1513 and 695 exhibited a complete, 340-bp sequence, and the other Orientalis isolates exhibited a 16-bp deletion; Medievalis isolates featured a 48-bp deletion and a mutation G → A at position 135 of the spacer; Antiqua isolates exhibited a 32-bp deletion except for isolate 611, which had a 16-bp deletion and mutations C164 → T, C166 → T and A191 → G. Spacer YP3 thus differentiated every biovar. As for spacer YP4, Medievalis isolates were characterized by a 36-bp deletion, whereas Antiqua and Orientalis isolates exhibited a complete spacer. For spacer YP5, Medievalis isolates exhibited a full-length 292-bp spacer, whereas Antiqua and Orientalis isolates exhibited a 32-bp deletion at position 101 of the spacer. For spacer YP7, a total of nine alleles were found; Antiqua isolates were classified within seven alleles, Medievalis isolates within four alleles, and Orientalis isolates within five alleles. Alleles two and three were found only among Orientalis isolates. A full-length sequence of 330 bp was found for strain 549; the other isolates exhibited deletions. At position 126 in the spacer, strains 643 and 695 exhibited a 56-bp deletion; at position 133, strains 304, 1092, 507 and 571 exhibited a 49-bp deletion; at position 140, strains 611, 685, and 537 exhibited a 42-bp deletion; at position 142, strains 616, 548, 565, 518, 520, 560, 561, and 670 exhibited a 35-bp deletion; at position 149, strains 519, 542, 566, 564, and 1513 exhibited a 28-bp deletion; at position 155, strains 552, 613, 772, and 989 exhibited a 21-bp deletion; at position 162, strains 550, 677, and 1594 exhibited a 14-bp deletion; and at position 169 in the spacer, strains 544, 553, and 545 exhibited a 7-bp deletion. As for spacer YP8, Antiqua and Medievalis isolates exhibited a full-length 236-bp spacer, whereas Orientalis isolates had an 18-bp deletion at position 36 of the spacer. As for spacer YP9, Antiqua and Medievalis isolates exhibited a full-length 292-bp sequence; Orientalis isolates had an 18-bp deletion located at position 123 of the spacer. As for YP10 spacer, Y. pestis CO92 strain had a unique sequence of 369-bp; all the other isolates exhibited an 18-bp deletion located at position 177 of the spacer. Sequences herein determined were deposited in GenBank (Table A1). Phylogenetic Analysis Among the 35 studied Y. pestis strains, we identified three main phylogenetic clusters, each of which included only strains from a single biovar, i.e., Orientalis, Medievalis, or Antiqua, as observed in the unrooted dendrogram (Figure 1). The same topology was obtained when data were analyzed by parsimony, neighbor-joining, and maximum likelihood analyses (Figure A3). Within the Medievalis cluster, strains 519 and 616, and strains 557 and 564 were grouped by pairs; no other subgroup was identified. Within the Orientalis cluster, three groups were identified; one group included strains 989, 613, 772, and 1513; a second group was made up of three pairs of strains, i.e., 304 and CO92, 507 and 571, and 685 and 1537; the third group diverged before the differentiation of the other two groups and contained strains 695 and 643. Within the Antiqua cluster, two groups were identified; one group included strains 553, 544, 545, 550, and 677, with the last two clustered; the second group comprised strains 552, 542, and 566; strains 548 and 549 did not group with either of these two groups but diverged before the differentiation of the other Antiqua strains. The Antiqua strain 611 exhibited a unique phylogenetic position. This strain differed from all other studied Y. pestis strains and appeared to have diverged before the separation of the three main clusters. Figure 1 Unrooted tree showing the phylogenetic relationships among the 35 studied Yersinia pestis isolates inferred from sequence analysis of the combination of the eight intergenic spacers using the unweighted pair group method with arithmetic mean method. O, Y. pestis Orientalis biovar; M, Y. pestis Medievalis biovar; A, Y. pestis Antiqua biovar. Numbers refer to the isolate number as in Table 1. MST of Ancient Dental Pulp Specimens In the 46 PCR experiments we performed on ancient tooth samples, we obtained 10 Y. pestis sequences (Figure 2); no sequences were found in the 51 PCR experiments with control teeth (p < 10–4). The teeth from 7 of the 8 persons' remains yielded 10 specific sequences, 3 persons were positive for two molecular targets, but none of the teeth of 17 persons used as negative controls yielded specific sequences (p < 10–4). YP1 PCR yielded an amplicon in one of six tested persons, YP8 PCR yielded an amplicon with identical sequence in six of six tested persons, and YP3 PCR yielded an amplicon in three of seven tested persons. When compared with GenBank database (Table A2), theYP1 sequence obtained in skeleton 5 yielded complete similarity with the homologous region in Y. pestis C092 strain over 390 positions and 99% sequence similarity with the homologous region in Y. pestis KIM strain over 174 positions; the YP8 sequence obtained in the six persons yielded 99% sequence similarity with homologous region in Y. pestis C092 strain over 178 positions and complete sequence identity with homologous region in Y. pestis KIM strain over 116 positions; the YP3 sequence obtained in skeletons 4 and 8 yielded complete sequence identity with that of homologous region in Y. pestis strain CO92 over 364 positions and 98% sequence similarity with homologous region in Y. pestis KIM strain over 206 positions; the YP3 sequence obtained in human remain 2 yielded a 98% similarity with homologous region in Y. pestis CO92 strain over 283 positions and a 95% similarity with homologous region in Y. pestis KIM strain over 162 positions. For each one of these 10 amplicons, further matches dropped to <90% sequence similarity over short sequences of 10 nt to 50 nt. When blasted to our local Y. pestis spacer sequence database, the YP1 spacer sequence obtained in human skeleton 5 was identical to that of the Orientalis and Antiqua reference sequences (Table A3), whereas smaller BLAST scores were obtained for the Medievalis reference sequences. Regarding the six YP8 spacer sequences obtained in skeletons 1–6, the 12 first best scores were obtained with Orientalis reference sequences. For the YP3 spacer sequence obtained in skeletons 4 and 8, the 10 first best scores were obtained with Orientalis reference sequences. For the YP3 spacer sequence obtained in skeleton 2, the 10 first best scores were obtained with Orientalis reference sequences, and this amplicon exhibited two specific nucleotide substitutions (Figure 2; Table A2). These mutations were consistently obtained in six clones. Figure 2 Molecular detection of Yersinia pestis was achieved in the dental pulp of remains of humans excavated from one Justinian and two Black Death mass graves in France by spacer amplification and sequencing (+, positive polymerase chain reaction [PCR] amplification and sequencing; –, absence of PCR amplification; ND, not done). Sequence analyses showed strains were of Orientalis genotype in all sets of remains; one of them exhibited two mutations numbered according to Y. pestis CO92 strain genome sequence (GenBank accession no. NC-003143). Negative control teeth remained negative. Discussion Our data show that MST differentiates the three biovars of Y. pestis in a collection of 35 isolates representative of the three biovars and originating from various sources and 13 countries. This finding suggests that MST data can be extrapolated to the entire Y. pestis species. Pulsed-field gel electrophoresis (PFGE) has been the only other technique that allows for biovar and strain differentiation, but it requires large amounts of cultured microorganisms, and the stability of PFGE profiles in subculture has been questioned ( 9 ). Ribotyping did not classify isolates into their respective biovars ( 9 , 21 , 22 ). Specific insertion sequences (IS), including IS100 ( 23 ), IS285 ( 24 , 25 ), and IS1541 ( 26 , 27 ), were used as markers in restriction fragment length polymorphism (RFLP) analyses ( 8 , 28 ) and in PCR-based technique ( 29 ). The last approach produced identical patterns of IS100 distribution in Antiqua and Medievalis isolates ( 29 ). A variable-number tandem repeat technique ( 30 , 31 ) had a greater discrimination capacity than did ribotyping, but isolates from different areas were found to harbor identical types. Sequencing of fragments of five housekeeping genes in 36 Y. pestis isolates from various locations and from 12 to 13 isolates from Y. pseudotuberculosis and Y. enterocolitica did not show diversity in any Y. pestis housekeeping gene ( 8 ). Indeed, 19 of these 36 Y. pestis isolates were also included in the present work and featured 12 different MST profiles, thus demonstrating the validity of our hypothesis and the usefulness of MST for Y. pestis genotyping. Morever, its format is applicable to microbial analyses of ancient samples since it requires small amounts of DNA and targets small genomic fragments. Few studies have aimed to disclose intraspecifc phylogenetic relationships of Y. pestis isolates. RFLP, probed with the IS100, indicated that, among 36 Y. pestis isolates, the three biovars formed distinct branches of the phylogenetic tree and that Y. pestis was a clone that evolved from Y. pseudotuberculosis 1,500–20,000 years ago ( 8 ). Isolates of biovars Antiqua and Medievalis clustered altogether apart from those belonging to biovar Orientalis. Likewise, a dendrogram constructed by the UPGMA clustering method on a PCR-based IS100 fingerprint database clearly discriminated Y. pestis isolates of the Orientalis biovar that formed a homogeneous group, whereas isolates of the Antiqua and Medievalis biovars mixed together ( 29 ). Isolates of biovar Antiqua showed a variety of fingerprinting profiles, whereas Medievalis isolates clustered with the Antiqua isolates originating from Southeast Asia, which suggests their close phylogenetic relationships. In this study, one isolate (strain Nicholisk 51) displayed a genotyping pattern typical of biovar Orientalis isolates, although this isolate was biovar Antiqua and lacked a 93-bp deletion within the glycerol-3-phosphate dehydrogenase glpD gene characteristic for the glycerol-negative Orientalis biovar. Our data support the view that most Y. pestis isolates cluster according to their biovar, but isolates of biovar Antiqua are more distantly related to other isolates than biovars Orientalis and Medievalis are. MST-based phylogenetic reconstructions unexpectedly found that one Y. pestis Antiqua isolate, number 611, formed a fourth branch, which suggests that Y. pestis may comprise four different lineages instead of the three that have been recognized so far. This unique isolate had not been included in Achtman and collaborators' study ( 8 ). MST was applied to ancient human specimens to test the hypothesis that three biovars were responsible for the three historical pandemics. Contamination of ancient samples by modern Y. pestis DNA and cross-contamination were prevented in our experiments. Indeed, we carried out two independent experiments, each with new reagents, in a laboratory where Y. pestis had never been introduced or studied, without positive controls ( 17 ). Both experiments produced consistent results, and negative controls were always negative. That we obtained a unique YP3 sequence further excludes the possibility of contamination, since this sequence differs from all the currently known sequences. This unique sequence was consistently found in six of six clones and thus did not result from the false incorporation of nucleotides by the DNA polymerase. In our previous work on the Black Death, we also reported a unique sequence ( 17 ). In the present study, the accurate identification of Y. pestis was confirmed by using two successive sequence analyses. We first blasted the sequences derived from ancient specimens against the GenBank database and observed best matches for Y. pestis homologous sequences, thus ensuring accurate species identification of the amplicons. We then blasted these sequences against our local Y. pestis spacer sequence database and found best matches for homologous sequence in Orientalis reference isolates for the three tested spacers. As our local database has 35 different reference sequences representative of the three Y. pestis biovars, there is no doubt regarding the identification nor the fact that Orientalis-like Y. pestis alone was implicated in the personal remains that we investigated. DNA of Y. pestis was recovered from remains of persons in one mass grave established to be of the Justinian pandemic era on the basis of radiocarbon dating (Figure A4). We found that the genotype Orientalis, which now occurs worldwide, was involved in all three pandemics. Also, we detected Y. pestis in additonal human remains from Black Death sites, which adds more evidence for its role in the second pandemic in southern France (four sites tested positive) ( 18 , 19 ). Indeed, historical descriptions were suggestive of bubonic plague in medieval southern Europe; in northern Europe, historical data indicated that the Black Death had a different epidemiologic pattern. This finding may indicate that latter outbreaks in the north were not caused by transmission of Y. pestis by blocked rat fleas but rather by mechanical transmission of plague bacteria by another ectoparasite that used humans as their primary hosts. Alternatively, another pathogen may have caused these outbreaks, and a search for Y. pestis in the dental pulps of suspected plague victims in Copenhagen (two persons' remains) and Verdun (five persons' remains) dating from the 18th century failed to show Y. pestis DNA ( 32 ). Further studies may elucidate the respective role of Y. pestis and other pathogens that may have contributed to deaths in these times.
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Modeling the epidemiological history of plague in Central Asia: Palaeoclimatic forcing on a disease system over the past millennium
Kyrre Kausrud, Mike Begon, Tamara Ari … (2010)
Background Human cases of plague (Yersinia pestis) infection originate, ultimately, in the bacterium's wildlife host populations. The epidemiological dynamics of the wildlife reservoir therefore determine the abundance, distribution and evolution of the pathogen, which in turn shape the frequency, distribution and virulence of human cases. Earlier studies have shown clear evidence of climatic forcing on contemporary plague abundance in rodents and humans. Results We find that high-resolution palaeoclimatic indices correlate with plague prevalence and population density in a major plague host species, the great gerbil (Rhombomys opimus), over 1949-1995. Climate-driven models trained on these data predict independent data on human plague cases in early 20th-century Kazakhstan from 1904-1948, suggesting a consistent impact of climate on large-scale wildlife reservoir dynamics influencing human epidemics. Extending the models further back in time, we also find correspondence between their predictions and qualitative records of plague epidemics over the past 1500 years. Conclusions Central Asian climate fluctuations appear to have had significant influences on regional human plague frequency in the first part of the 20th century, and probably over the past 1500 years. This first attempt at ecoepidemiological reconstruction of historical disease activity may shed some light on how long-term plague epidemiology interacts with human activity. As plague activity in Central Asia seems to have followed climate fluctuations over the past centuries, we may expect global warming to have an impact upon future plague epidemiology, probably sustaining or increasing plague activity in the region, at least in the rodent reservoirs, in the coming decades. See commentary: http://www.biomedcentral.com/1741-7007/8/108
Author and article information
Journal
Journal ID (nlm-ta): Clujul Med
Journal ID (iso-abbrev): Clujul Med
Journal ID (publisher-id): CM
Title:
Clujul Medical
Publisher:
Iuliu Hatieganu University of Medicine and Pharmacy
ISSN
(Print):
1222-2119
ISSN
(Electronic):
2066-8872
Publication date
(Print):
2015
Publication date
(Electronic):
15
November
2015
Volume: 88
Issue: 4
Pages: 563-566
Affiliations
[1
]Department of Fundamental Disciplines and Clinical Prevention, Faculty of Medicine,
Transilvania University of Brasov, Romania
Author notes
Address for correspondence:
ddumitrascu@
123456umfcluj.ro
Article
Publisher ID: cm-88-563DOI: 10.15386/cjmed-546
PMC ID: 4689253
SO-VID: 702c9fe1-ba58-45af-a050-555e5a720cbe
Copyright statement:
Copyright @
2015
License:
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License
History
Date
received
: 20
July
2015
Date
accepted
: 07
August
2015
Categories
Subject:
History of Medicine and Pharmacy
Data availability: