Concepts of macro- and microevolution as related to the problem of origin and global expansion of the plague pathogen Yersinia pestis

The relationship between macro- and microevolutionary processes is considered with reference to the ecological scenario of the origin of the plague pathogen and its subsequent natural and anthropogenic global expansion. The macroevolutionary transformation of the ancestral pseudotuberculosis microbe clone into the initial plague microbe Yersinia pestis tarbagani occurred in Central Asia at the end of the Late Pleistocene by a “vertical” Darwinian way in an inadaptive heterothermal continual intermediate environment—the Mongolian marmot Marmota sibirica—flea Oropsylla silantiewi system—via a sequence of unstable and currently extinct intermediate forms. Its natural geographic expansion on the “oil spot” principle in the postglacial time led to the microevolutionary formation of 20–30 hostal subspecies circulating in populations of the background species of burrowing rodents and pikas in arid areas of Eurasia. The intercontinental spread of the “marmot” and “rat” pathogen subspecies in the past few centuries has been exclusively anthropogenic, with the involvement of synanthropic (ship) rats.     

Changes in Flea (Siphonaptera) Vector Activity in the Siberian Natural Plague Foci

Comparative analysis of vector activity of fleas in the Siberian natural plague foci was carried out during two long-term periods of experimental studies: 1967–1980 and 1983–2007. The data on block formation frequency in adult fleas infected with Yersinia pestis were analyzed for 127 experiments with 15 flea species and subspecies. The vector activity of fleas in all the Siberian plague foci (Altai, Tuva, and Transbaikalia) has increased over a rather short time period of 30–40 years. The frequencies of flea blocking were significantly different (P < 0.001) between the analyzed periods in all the three plague foci.

     

Ecological aspects of the origin of Yersinia pestis,causative agent of the plague: Concept of intermediate environment

Modern phylogenies of Yersinia pestis (Logh.), causative agent of the plague, constructed using molecular-genetic methods, do not receive a satisfactory functional and adaptive interpretation and are far from being ecologically valid. We have presented an ecological scenario of the origin of the causative agent of the plague through the transition of the initial pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b to a free hostal ecological niche (and a new adaptive zone) under ultracontinental climatic conditions of the Late Pleistocene (Sartan time, 22000–15000 years ago) in southern Siberia and Central Asia. An intermediate environment, i.e., the “Mongolian marmot Marmota sibirica-flea Oropsylla silatiewi” parasitic system, where the process of adaptation development of the plague microbe took place, has been characterized. A scenario based on the major principles of the modern synthetic theory of evolution opens the way to an ecological-genetic synthesis of the problem of plague origin and is an appropriate model for developing a theory of molecular evolution of pathogenic (plaguelike) microorganisms.     

Sympatric speciation of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Monohostal specialization in the host–parasite marmot–flea (<Emphasis Type="Italic">Marmota sibirica–Oropsylla silantiewi</Emphasis>) system

An ecological scenario of the origin of the plague microbe that is interpreted in the light of modern Darwinism (synthetic theory of evolution) is presented. It is shown that the plague microbe emerged from a clone of the psychrophilic saprozoonotic pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan time, 22000–15000 years ago, in the monohostal Mongolian marmot (Marmota sibirica)–flea (Oropsylla silantiewi) host–parasite system. It was noted that the evolutionary process described corresponds to the sympatric form of speciation by transition of the clone of migrant founders to a new, already-existing ecological niche. It was established that monohostal specialization of the plague microbe was made possible due to heterothermia (5–37°C) of marmots in the hibernation period. The factors of the speciation process—isolation, the struggle for existence, and natural selection—were analyzed.     

Results of using the serological method of epidemiological investigation of plague foci in the Muyunkum and Eastern Kyzylkum deserts in 1978-1982

Results of serological examination of 81,853 mammals from the Muyunkum and 26,303 from the Eastern Kyzylkum deserts were compared with results of a corresponding bacteriological examination for plague. In both foci and in all phases throughout the epizootic of plague, the serological method made it possible to detect infected animals 6-10 times more frequently in comparison with the bacteriological method. The big meriones mice, i.e., the basic carriers of plague in natural foci, were found to be infected considerably more (in percentage) frequently than the meridian meriones. Differences in the reactivity of meriones to the invasion of plague microbe were observed in both foci (the indices of titres in the antigen neutralization reaction were considerably higher in the Eastern Kyzylkums). The serological method of investigation is highly informative in evaluating both the present state of epizootic activity of a plague focus and its activity in the past.     

Broader investigation into the external environment of the specific antigen of the infectious agent in epizootiological observation and study of the structure of natural foci of plague.

The possibilities of detection of the infectious agent in natural foci of plague can be expanded to some extent by using the method of search for the specific FI capsular antigen of the plague microbe in such objects of the external environment which have not yet been subjected to laboratory analysis and serological analysis in particular. Such objects include soil from the burrow and the substrate of the nest of the rodent as well as the fleas of rodents and regurgitations of birds of prey.The effectivity and suitability of examination of these objects using immuno-adsorption reactions with standard erythrocyte diagnostics (passive haemagglutination reaction, antibody neutralization reaction) were demonstrated experimentally and confirmed under field conditions in various geographical and ecological zones of the Caucasus. Thus in 1971, an earlier unknown natural focus of plague was found in the Mount Elbrus massif by means of serological analysis of the substrate of the nest and of the soil from the burrows of rodents. It can be expected that broadened serological search for the FI antigen of plague will prove useful in the study of the structure of natural foci of plague, in the reconnaissance or retrospective investigation of poorly explored territories and in the solution of some questions concerning the epizootiology of plague, such as the survival of the infectious agent in the inter-epizootic period, telluric plague, etc.     

Genetic bases of methionine dependence in <Emphasis Type="Italic">Yersinia pestis</Emphasis> strains of main and non-main subspecies

Structural and functional organization of genes responsible for biosynthesis of amino acid methionine, which plays a leading role in cellular metabolism of bacteria, was studied in 24 natural Yersinia pestis strains of the major and non-main subspecies from various natural plague foci located in the territory of Russian Federation and neighbouring foreign countries, and also in Y. pestis and Y. pseudotuberculosis strains recorded in the files of NCBI GenBank database. Conservatism of genes metA, metC, metE, and metH as well as regulatory genes metR and metJ involved in biosynthesis of this amino acid was established. Sequencing of the variable locus of gene metB in natural Y. pestis strains of major and non-main subspecies revealed that the reason for the methionine dependence of strains belonging to the main subspecies is a deletion of a single nucleotide (−G) in the 988 position from the beginning of the gene, whereas this dependence in strains belonging to subspecies hissarica results from the appearance of a single nucleotide (+G) insertion in the 989 position of gene metB. These mutations are absent in strains of the caucasica, altaica, and ulegeica subspecies of the plague agent and in strains of pseudotuberculosis microbe, which correlates with their capacity for methionine biosynthesis.     

Exploration of natural foci of tularemia and plague in Armenia using the serological examination of bird droppings and excrements of predatory mammals]

Forty strains of tularemia and 619 of plague microbes were isolated in 1974 in bacteriological examination of natural plague and tularemia foci from a great number of small mammals and their ectoparasites. At the same time in serological examination (in the antibody neutralization test) of bird pellets, 52 mummified cadavers, and 34 excretion samples of mammalian beasts of prey collected in Armenia (its central and North-Western part) in 1973 the antigen of tularemia microbe was revealed in 73, 8, and 3, and of plagye--in 42, 5, and 1 cases, respectively. In one of the sites examined the number of positive findings failed to exceed 10--17%, this indicating a low intensity of the epizootic in the majority of the places. Judging by the mean titres of the serological test, which varied from 1:12 to 1:1428 in examination for tularemia and from 1:12 to 1428 in examination for plague, it was possible to detect both epizootics coursing during the examination, and those which occurred several monts ago. Tularemia and plague foci were not infrequently present at the same territories, and these diseases could involve the same individual animals of Microtus arvalis (Pall.). The data obtained pointed to the greater effectiveness of examination of bird's pellets and excretions of mammalian beasts of prey for the reconnaisance investigation of the natural foci of plague and tularemia in comparison with the bacteriological methods applied usually.     

Origin of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Structure of the process of speciation

The origin and evolution of the plague microbe Yersinia pestis are considered in the context of propositions of modern Darwinism. It was shown that the plague pathogen diverged from the pseudotuberculous microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan: 22000-15000 years ago. Speciation occurred in the tarbagan (Marmota sibirica)-flea (Oropsylla silantiewi) parasitic system. The structure of the speciation process included six stages: isolation, genetic drift, enhancement of intrapopulational polymorphism, the beginning of pesticin synthesis (genetic conflict and emergence of hiatus), specialization (stabilization of characteristics), and adaptive irradiation (transformation of the monotypic species Y. pestis tarbagani into a polytypic species). The scenario opens up wide prospects for construction of the molecular phylogeny of the plague microbe Y. pestis and for investigation of the biochemical and molecular-genetic aspects of “Darwinian” evolution of pathogens from many other nature-focal infections.     

The phenomenon of <Emphasis Type="Italic">Yersinia pestis</Emphasis> biofilm formation in the organism of fleas

The paper considers, for the first time, the formation of the extracellular matrix envelope (EME), or the biofilm, by Yersinia pestis as the basis determining the nature of interaction of the plague agent with the flea organism. The significance of the insect proventriculus in the process of biofilm formation is shown. The ultrastructure of the conglomerates of the plague microbe in the flea proventriculus and midgut was studied and the uniform mechanism of their formation was established. The role of Yersinia pestis biofilm in preservation of the plague microbe in the intestine of ectoparasites and in the soil of rodent burrows was discussed. PCR analysis confirmed the presence of the agent in plague infected corpses and flea feces stored at +8−10°C for 7 years and 9 months.     

Development of views on natural focality of plague

The theory of natural focality of human diseases, basically formulated by Academician E.N. Pavlovsky in 1939, invigorated research on plague, the disease whose epidemics had taken millions of lives in the past. Numerous studies in Russia and abroad have provided a great amount of data on the pathogen, its carriers, and vectors, specific features of infection spread and dynamics of activity of natural plague foci. Over a long period, plague was considered to be an obligate transmissible zoonosis. However, recent laboratory experiments and direct observations in natural foci of this infection indicate that plague is probably a zoophilous sapronosis.     

Dynamics of Yersinia pestis and Its Antibody Response in Great Gerbils (Rhombomys opimus) by Subcutaneous Infection

Background

Rhombomys opimus (great gerbil) is a reservoir of Yersinia pestis in the natural plague foci of Central Asia. Great gerbils are highly resistant to Y. pestis infection. The coevolution of great gerbils and Y. pestis is believed to play an important role in the plague epidemics in Central Asia plague foci. However, the dynamics of Y. pestis infection and the corresponding antibody response in great gerbils have not been evaluated. In this report, animal experiments were employed to investigate the bacterial load in both the liver and spleen of infected great gerbils. The dynamics of the antibody response to the F1 capsule antigen of Y. pestis was also determined.

Methodology

Captured great gerbils that tested negative for both anti-F1 antibodies and bacterial isolation were infected subcutaneously with different doses (10⁵ to 10¹¹ CFU) of a Y. pestis strain isolated from a live great gerbil during routine plague surveillance in the Junggar Basin, Xinjiang, China. The clinical manifestations, changes in body weight, anal temperature, and gross anatomy of the infected animals were observed. The blood cell count, bacterial load, and anti-F1 antibody titers were determined at different time points after infection using a blood analyzer, plate counts, and an indirect hemagglutination assay, respectively.

Conclusions/Significance

The dynamics of bacterial load and the anti-F1 antibody concentration in great gerbils are highly variable among individuals. The Y. pestis infection in great gerbils could persist as long as 15 days. They act as an appropriate reservoir for plague in the Junggar Basin, which is part of the natural plague foci in Central Asia. The dynamics of the Y. pestis susceptibility of great gerbil will improve the understanding of its variable resistance, which would facilitate the development of more effective countermeasures for controlling plague epidemics in this focus.     

Principles of speciation of the plague causative agent <Emphasis Type="Italic">Versinia pestis</Emphasis>: Gradualism or saltation?

The saltation origin of the causative agent of the plague Yersinia pestis from the pseudotuberculosis microbe Y. pseudotuberculosis O:1b has been proclaimed in recent investigations on molecular genetics. The speciation process in this case is proposed to be connected with horizontal transfer of exogenous genetic structures (such as specific plasmids pFra and pPst) into the genome of the ancestral form. The alternative “Darwinian” model of the gradual origin of the plague agent is proposed based on ecological factors. The comparison of two evolutionary scenarios, saltation and gradual, is performed; the latter seems more likely.     

HOST-SPECIFICITY AND HOST-SELECTION OF FLEAS IN FOCI OF HUMAN PLAGUE IN YUNNAN CHINA*

Abstract The host-specificity and the host-selection of 11 species of fleas collected from 47 species of small mammals in foci of human plague in Yunnan, China, were studied by using methods in the evaluation of ecological niche breadth and overlap. Levins' niche breadth was used for the host-specificity. while clip angle niche overlap and a fuzzy clustering analysis were used for host-selection. Of the 11 species of fleas, the host-specificity of Nosopsyllus elongatus puerensis and Xenopsylla cheopis are the highest (narrow niche breadth), and those of Aviostivalis klossi bispiniformis and Neopsylla stevensi sichuanyunnana the lowest (wide niche breadth). Of 11 species of fleas, the dominant host of X. cheopis (a very high effective vector of plague in the foci of human plague in Yunnan Province. China) is Rattw flavipectus (the main animal host and infectious source of plague in the foci). A high host-specificity of X. cheopis implies that X. cheopis mainly maintains or transmits the pathogen of plague among the individuals of its dominant species of host, R. flavipectus. The result of niche overlap analysis reveals that Ctenophthalrnus In-evipre jiciens and Ctenophthaltnus parcus have a similar host-selection while other species of fleas are quite different in their host selection.     

Analysis of insect toxin complex gene variability of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> strains

The nucleotide sequences of the Tc’s insect toxin complex genes have been analyzed in 18 natural strains of the main and non-main subspecies of Yersinia pestis isolated in different natural foci in the Russian Federation, as well as neighboring and more remote countries, as compared to the data on Y. pestis and Y. pseudotuberculosis strains stored in the NCBI GenBank database. The nucleotide sequences of these genes in plague agent strains have been found to be highly conserved, in contrast to those of the pseudotuberculosis agent. The sequences of two genes, tcaC and tccC2, have been found to be almost identical in Y. pestis strains, whereas other three genes (tcaA, tcaB, and tccC1) contain a few mutations, which, however, are not common for all strains of the plague agent. Exceptions are only strains of the Y. pestis biovar orientalis, whose tcaB gene is in a nonfunctional state due to a nucleotide deletion. The results suggest that the formation of the species Y. pestis as an agent of a natural focal infection with a transmissive mechanism has not resulted in degradation of the Tc’s complex genes. Instead, these genes are likely to have been altered as the plague agent have been adapting to the new environment.     

Yersinia pestis strains isolated in natural plague foci of Caucasus and Transcaucasia in the context of the global evolution of species

BackgroundPlague is a highly dangerous vector-borne infectious disease that has left a significant mark on history of humankind. There are 13 natural plague foci in the Caucasus, located on the territory of the Russian Federation, Azerbaijan, Armenia and Georgia. We performed whole-genome sequencing of Y. pestis strains, isolated in the natural foci of the Caucasus and Transcaucasia. Using the data of whole-genome SNP analysis and Bayesian phylogeny methods, we carried out an evolutionary-phylogeographic analysis of modern population of the plague pathogen in order to determine the phylogenetic relationships of Y. pestis strains from the Caucasus with the strains from other countries.ResultsWe used 345 Y. pestis genomes to construct a global evolutionary phylogenetic reconstruction of species based on whole-genome SNP analysis. The genomes of 16 isolates were sequenced in this study, the remaining 329 genomes were obtained from the GenBank database. Analysis of the core genome revealed 3315 SNPs that allow differentiation of strains. The evolutionary phylogeographic analysis showed that the studied Y. pestis strains belong to the genetic lineages 0.PE2, 2.MED0, and 2.MED1. It was shown that the Y. pestis strains isolated on the territory of the East Caucasian high-mountain, the Transcaucasian high-mountain and the Priaraksinsky low-mountain plague foci belong to the most ancient of all existing genetic lineages - 0.PE2.ConclusionsOn the basis of the whole-genome SNP analysis of 345 Y. pestis strains, we describe the modern population structure of the plague pathogen and specify the place of the strains isolated in the natural foci of the Caucasus and Transcaucasia in the structure of the global population of Y. pestis. As a result of the retrospective evolutionary-phylogeographic analysis of the current population of the pathogen, we determined the probable time frame of the divergence of the genetic lineages of Y. pestis, as well as suggested the possible paths of the historical spread of the plague pathogen.     

Study of trimethoprim sensitivity in strains of Yersinia pestis isolated from various natural foci of plague

The effect of exogenic addition of purines and pyrimidines on trimethoprim sensitivity was studied in 134 vaccinal and virulent strains of the plague causative agent isolated at various periods in 12 natural plague foci. 74 strains proved to be sensitive to low concentrations of trimethoprim in the cultivation media irrespective of the presence or absence of thymine or thymidine in them. In this respect the strains differed from many other bacterial species which in the presence of thymidine or thymine were resistant to high concentrations of trimethoprim. 60 natural arginine deficient strains of the plague causative agent from Transcaucasia and Mongolia showed high levels of resistance to trimethoprim on media with thymine or thymidine. The possible mechanism of the plague microbe sensitivity to trimethoprim in the presence of thymine or thymidine is discussed with an account of the literature and original data.     

Yersinia enterocolitica O9 as a Possible Barrier Against Y. pestis in Natural Plague Foci in Ningxia, China

A survey of Yersinia spp, as related to plague control, was made in Haiyuan of Ganning loess plateau plague focus, Yanchi of Inner Mongolia plateau plague focus, and Yinchuan city, as a control area, in Ningxia, China. In Haiyuan, where the main plague reservoir was Mongolian ground squirrel (Citellus alaschanicus) living in the prairie, Y. enterocolitica O9 was frequently isolated from pigs, dogs, rodents living in and around houses, but only rarely from hare and Mongolian ground squirrel. In Yanchi, where the main plague reservoir was Mongolian gerbil (Meriones unguiculatus) living in the prairie and Y. pestis, which was isolated from rodents up to 1991, Y. enterocolitica O9 was sometimes isolated from pigs and rodents. In all areas, some strains of Y. enterocolitica O3 and Y. pseudotuberculosis serotypes 3 and 4b were also isolated from pigs, dogs, and from rodents. We propose that an epidemiological link exists between the prevalence of Y. pestis and Y. enterocolitica O9 in domestic and rodents living in these areas in China. The residential area in Haiyuan may be protected against Y. pestis by the domestic animals and rodents which acquired cross-protection against Y. pestis by infection with Y. enterocolitica O9, but this is not the case in the Yanchi district. Received: 14 February 2000 / Accepted: 17 July 2000     

Plague studies in California: a review of long‐term disease activity,flea‐host relationships and plague ecology in the coniferous forests of the Southern Cascades and northern Sierra Nevada mountains

We review 28 years of long‐term surveillance (1970–1997) for plague activity among wild rodents from ten locations within three coniferous forest habitat types in the northern Sierra Nevada and the Southern Cascade mountains of northeastern California. We identify rodent hosts and their fleas and document long‐term plague activity in each habitat type. The highest seroprevalence for Yersinia pestis occurred in the chipmunks, Tamias senex and T. quadrimaculatus, and the pine squirrel, Tamiasciurus douglasii. The most commonly infected fleas were Ceratophyllus ciliatus and Eumolpianus eumolpi from chipmunks and Oropsylla montana and O. idahoensis from ground squirrels. Serological surveillance demonstrated that populations of T. senex, T. quadrimaculatus and T. douglasii are moderately resistant to plague, survive infection, and are, therefore, good sentinels for plague activity. Recaptured T. senex and T. quadrimaculatus showed persistence of plague antibodies and evidence of re‐infection over a two year period. These rodent species, their fleas, and the ecological factors common to the coniferous forest habitats likely promote the maintenance of plague foci in northeastern California.     

Efficiency of Neopsylla abagaitui fleas as plague vectors in Transbaikalia

Experiments have shown that the block of proventriculus develops in 1.1 to 1.5% of individuals of the flea Neopsylla abagaitui infected with plague microbe. These insects transmit the agent during bloodsucking to different plague carriers (Citellus dauricus, C. undulatus, Meriones unguiculatus, Microtus gregalis). The plague microbe is preserved in fleas for 65 days (the observation period).