Relationship of the flea Citellophilus tesquorum altaicus (Siphonaptera: Ceratophyllidae) with the plague agent

Some aspects of relationships of the flea Citellophilus tesquorum altaicus and bacterium Yersinia pestis of two strains isolated from different parts of the Tuva natural plague focus were studied. Peculiarities of elimination and blood meal activity of fleas infected with two strains of the plague agent were not revealed. Differences in mortality and alimentary activity are considerably determined by the sex of insects. The ability of examined strains to form a proventriculus block was not identical in the strains examined. This ability was expressed higher in the strain I-3428, which originated from the same part of the natural focus as the insectarium flea culture, than in the strain I-3327. During the spring and first half of summer, the proventriculus block appeared more frequently in females. The increasing of the fraction of blocked individuals was observed in both sexes from spring to summer. As for the ability to transmit the plague agent, similar seasonal increasing was noted in males, but in females, the ability to inoculate the plague microbe was always maintained at the same level.     

Changes in the numbers of the causative agent of intestinal yersiniasis (Yersinia enterocolitica) in Xenopsylla cheopis (Aphaniptera) fleas in the process of blood digestion]

In infected fleas the agent of intestinal yersiniosis underwents a complex cycle of quantitative changes after each feeding. A species belonging of blood consumed greatly affected the dynamics of the agent abundance. The general peculiarity of the development of microbes in insects, which fed on various animals (white mice, Sirian hamsters, white rats and guinea pigs), is characterized by the decrease in the abundance of the agent during the first hours after feeding. This was followed by an active multiplication of microbes replaced by a new fall after which the abundance maintained on the level close to the initial one. A comparison of obtained results with the data on the digestion in fleas has shown that the phases of the primary dying off and depression of the agent falls within the intensive-decay of the food clot. The active multiplication proceeds at the end of digestion that may be promoted by the decrease in the fermentative activity and abundance of products of blood decay easily assimilated by microbes. The next fall in the agent's abundance and the absence of multiplication are associated with the exhaustion of the nutrient medium in the process of absorbtion and vital activity of microbes.     

Characteristics of the multiplication of a virulent strain of the plague microbe in Xenopsylla cheopis fleas infected parenterally

Xenopsylla cheopis fleas infected parenterally with the virulent strain of plague microbe of gerbil variant preserved the agent to the end of their lives. In the body cavity the microbes retained their ability for reproduction which was, however, limited. During the first seven days after the infection the number of microbes slightly increased and later became stabilized. Its mean indices (mean g) varied within the limits of 500 to 2000 microbe cells per 1 individual, maximum index rarely exceeded 30 000 microbe cells. Parenteral infection with plague agent did not affect essentially the longevity of fleas.     

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.     

The epizootiological significance of flea accumulation in aggregations of the female long-tailed suslik in the Tuva plague focus

The formation of aggregations of related females is a peculiarity of the intrapopulational organization of long-tailed suslik that results in the accumulation of Citellophilus tesquorum, the main vector and keeper of the plague microbe in the Tuva plague nidus. The plague agent is adapted to the existence on the territory occupied by aggregations of females that manifests itself in the delay of the beginning and prolixity of block-formation periods in fleas. The transfer of the agent from one place to another takes place only at the high abundance of long-tailed suslik. When the number of the suslik is low the plague microbe circulates in the ranges of one or several neighbouring aggregations of females having no possibility for successful transfer to a new place.     

Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis]

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.     

Paradoxopsyllus scorodumovi (Aphaniptera) flea, an effective vector of plague in a Gorno-Altai natural focus

A study was conducted on the fleas of P. scorodumovi and five local strains of the plague microbe, one of which is typical of the strains of the Altai subspecies and four are non-typical of this nidus. The fleas of this species are capable to transmit not only the plague agent of the strains typical of this nidus but also non-typical ones which differ in some biological properties and are avirulent for most carriers but Pallas's pika. Biological peculiarities of fleas of P. scorodumovi in addition to their high efficiency as vectors of the plague microbe enable us to associate the more active autumn epizooty with fleas of this species.     

Iron assimilation by Yersinia pestis on iron-deficient media

The growth of plague bacteria may be limited by the level of iron concentration in the nutrient medium. The virulent strains of the plague microbe possess the more pronounced mechanism of iron assimilation as compared to the vaccine strain. The iron ions are extracted by the virulent and vaccine strains only under the cell surface contact with the iron-saturated transferrin. The iron-sorbing function is peculiar to the plague microbe cell walls which is pronounced more strongly in the virulent strains.     

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).     

Experimental evaluation of prospects for the use of beta-lactams in plague infection caused by pathogens with plasmid resistance to penicillins]

High therapeutic efficacies of ceftriaxone, ceftazidime, cefotaxime and azthreonam in the treatment of experimental plague induced by beta-lactamase-producing strains of the plague microbe containing R plasmids RP-1, R57b and R40a were shown to correlate with their in vitro antibacterial activities. The therapeutic efficacy of sulbactam/ampicillin was recorded in the treatment of plague induced by the strain containing R plasmids R57b and R40a (the treatment course of 7 days). However, it was lower when the infection was due to the strain containing plasmid RP-1 (beta-lactamase TEM-2). Cefoperazone was not active in the treatment of experimental plague induced by the strains containing plasmids RP-1 and R57b (beta-lactamases TEM-2 and OXA-3). Ceftriaxone versus the antibiotics tested was considered to be the drug of choice for the etiotropic therapy of plague induced not only by the type strains of the plague microbe but also by its variants with the plasmid pattern resistance to penicillins.     

Persistence of Francisella tularensis McCoy et Chapin tularemia agent in the organism of highly sensitive rodents after oral infection

In the literature there are no data on the possibility of obtaining in experiment non-fatal tularemia infection (persistence) in rodents highly sensitive to it (Group I) when using highly virulent strains circulating in nature for infection by natural routes. Our detailed experiments on 1483 adult voles Microtus rossiaemeridionalis Ogn. (syn. M. subarvalis Meyer et al.) of laboratory origin using virulent strains of Francisella tularensis holarctica Ols. et Meshch. and natural alimentary infection by feeding on bodies of died animals or forced dosed administration of a mixture of dead and living bacteria to the voles through the oesophagus demonstrated the possibility of the animals to survive tularemia with subsequent long-term chronic carrier state of the infectious agent. They also confirmed the ability of voles to eat readily cadavers of their kin (cannibalism, necrophagia). Experiments with the fully virulent strain 503 and feeding on cadavers were carried out on 439 voles. 203 animals died from acute tularemia, 43 from side effects and 193 survived. Two of the latter (0.5%) exhibited chronic bacterial carrier state, and agglutinins to tularemia microbe (1:320) were found in their blood. From 309 voles subjected to dosed feeding, 153 died from acute tularemia, 27 from side effects and 129 survived. Two of them were bacterial carriers and 6 (1.9%) showed agglutinins (1:160-1:1280). In experiments with strain 165, spontaneously less virulent for guinea pigs, 433 voles were fed on cadavers. 170 of them died from acute tularemia, 53 from side effects, and 210 animals survived. Among the latter, 14 animals (3.2%) were found immune to 100 LD50 of the highly virulent strain 1298. In dosed feeding of 302 voles with the strain 165, 90 animals died from acute tularemia, 59 from side effects, and 153 survived, including 63 animals (20.7%) immune to 100 LD50. The surviving immune voles exhibited seroconversion and long-term persistence of the infectious agent in the internal organs (up to day 257-313--period of observation), accompanied bacteriuria in some cases. Histological examination of the kidney revealed, for the first time, important pathological changes of glomerulonephritis type with elements of pyelonephritis. Protracted stay of the agent in the organism of the vole does not affect its virulence. Persistence of tularemia agent in the organism of voles highly sensitive to tularemia in alimentary administration to them of living and dead bacteria is achieved as a result of anticipatory development of immunological reactions in response to a massive dose of killed antigen, against the background of which the accumulation of simultaneously administered     

The epizootiological role of the population organization of the stock of fleas on the long-tailed suslik in a natural focus of plague in Tuva

Spatial location of epizootic events in the Tuva plague focus is determined at a considerable degree by the population structure of the flea Citellophilus tesquorum--the main plague microbe vector. Within the enzootic territory occupied by five populations of the long-tailed ground squirrel (Citellus undulatus) there are six populations of C. tesquorum. Each population of fleas has a corresponding autonomic plague focus. Various conditions for the microbe life activity in these populations are recovered. The circulation of the microbe is closely connected with certain intrapopulation groupings of fleas--the nuclei of populations.     

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.     

Sensitivity of different strains of the causative agent of plague to disinfectants]

Various strains of the plague microbe was found to have statistically significant differences in their sensitivity to phenol, formaldehyde and chloramine. These differences did not correlate with the origin, virulence and nutritional requirements of the plague strains. The sensitivity of the plague microbe to formaldehyde and phenol was found to be related to the permeability of its cell wall.     

Characteristics of digestion of blood of different animals by the flea Xenopsylla cheopis]

Under experimental conditions fleas X. cheopis engorged and assimilated blood of 4 species of rodents, man, pigeon and two species of reptiles. Histological investigations have shown that the structure of food clot, destruction rate of blood cells and general duration of digestion are changed considerably depending on the host's blood. In females the digestion of white mice blood to haematin at 23-24 degrees lasted not over 12 hours, the digestion of blood of hamsters and man - 12 to 20 hours, of guinea pigs and reptiles - 18 to 25 hours and of white rats and pigeons - from 20 to 30 hours. In males the digestion carries out somewhat slower. The above differences in blood digestion can apparently create unequal conditions for pathogenic microbes in the intestine of fleas that affects the preservation and reproduction of the agent in the organisms of these carriers.     

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.     

Yersinia--flea interactions and the evolution of the arthropod-borne transmission route of plague

Yersinia pestis, the causative agent of plague, is unique among the enteric group of Gram-negative bacteria in relying on a blood-feeding insect for transmission. The Yersinia-flea interactions that enable plague transmission cycles have had profound historical consequences as manifested by human plague pandemics. The arthropod-borne transmission route was a radical ecologic change from the food-borne and water-borne transmission route of Yersinia pseudotuberculosis, from which Y. pestis diverged only within the last 20000 years. Thus, the interactions of Y. pestis with its flea vector that lead to colonization and successful transmission are the result of a recent evolutionary adaptation that required relatively few genetic changes. These changes from the Y. pseudotuberculosis progenitor included loss of insecticidal activity, increased resistance to antibacterial factors in the flea midgut, and extending Yersinia biofilm-forming ability to the flea host environment.     

Doxycycline in the prevention of experimental plague induced by plague microbe variants]

A comparative study was performed on the efficacy of doxycycline in experimental plague infection induced in albino mice by strain 231 of the plague microbe and its variant 231 Fra- deprived of the ability to produce the fraction I antigen. It was shown that the LD50 for strain 231 during animal treatment with doxycycline was significantly higher than that for variant 231 Fra-. Prophylaxis of the plague infection caused by the Fra- forms of the plague microbe required significantly higher doses of doxycycline (ED50) than that of the infection caused by the Fra+ forms. The use of the daily maximum permissible doses of doxycycline (50 to 100 mg/kg a day) for 10 days in treatment of albino mice infected with the strain Fra- did not provide animal survival at the level higher than 60 to 70 per cent while the survival rate in the animals infected with the strain Fra+ of the plague microbe and treated according to the same scheme amounted to 90-100 per cent. The lower therapeutic efficacy of doxycycline in the treatment of the infection caused by the fractionless variant of the plague microbe should be considered in development of rational schemes for prophylaxis and treatment of plague.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

The efficiency of Rhadinopsylla rothschildi and R. dahurica fleas as vectors of the causative agent of plague in a Transbaikal natural focus

Experiments have shown that a block of proventriculus arises in 2.1 to 12.5% of infected fleas of R. rothschildi and in 7.2 to 10.5% of R. dahurica. These fleas transmit the plague agent to different animals (Brandt's vole, narrow-skulled vole, Dahurian suslik). The plague microbe is preserved in the organism of infected insects till the end of the experiment, 74 and 24 days respectively. Therefore, the fleas can play a part in the maintenance of plague epizootics in the Transbaikal natural nidus.