The formation of proventriculus block, alimentary activity and mortality of flea Amphipsylla primaris primaris infected with Yersinia pestis

The results of experiments held in 1982-1983 in Tuva plague natural focus with flea Amphipsylla primaris primaris (Jordan et Rothschild, 1915) from natural populations, whish were inflected and fed on specific host--flat-headed vole (Alticola strelzovi), are analyzed. The initial infectivity of the insects in autumn was higher than in spring: 90 and 50 % respectively. Accumulation of the agent in aggregated form in the organism of A. p. primaris, estimated by the quantity of fleas with and partial blocks, was more active in imago of both sexes in autumn than in spring, while sucking flea were observed in spring more often than in autumn. Irrespective of season, the part of males with visible accumulations of Y. pestis was more, and their alimentary activity was higher than that of females. Fleas died much more quickly in spring. Part of the males with proventriculus block exceeded that of females in spring experiment. Females with alimentary canal obstruction prevailed in autumn. Thus, sex of the insect and season of the experiment conducting influenced on all studied indices. Besides that, Y pestis ability for the proventriculus block formation in fleas during different seasons can change by the opposite way depending on sex of the ectoparasites.     

Seasonal dynamics of infecting ability of the flea Citellophilus tesquorum altaicus in the Tuva natural focus of the plague

The infecting ability of the fleas Citellophilus tesquorum altaicus loff, 1936, the main plague vectors in the Tuva natural focus, was experimentally studied in different periods of the epizootic season. Seasonal dynamics in the efficiency of infecting the long-tailed Siberian souslik with the plague causative agent through flea bites was noticed. Seasonal differences in infectivity of the "blocked" flea bites are revealed. An increase of infected experimental animals with a generalization of infection process in the period of epizooty activation in the natural focus was observed. A resistance of the long-tailed Siberian souslik to the plague agent infection through flea bites in the spring season was registered.     

Seasonal dynamics of epizootic process of the plague agent <Emphasis Type="Italic">Yersinia pestis</Emphasis> transmission to the long-tailed suslik <Emphasis Type="Italic">Citellus undulatus</Emphasis> by the flea <Emphasis Type="Italic">Citellophilus tesquorum</Emphasis> in Tuva

Transmission of Yersinia pestis to the long-tailed suslik (Citellus undulatus) by fleas (Citellophilus tesquorum) in the Tuva natural plague focus in different seasons (spring, summer, and autumn) was studied experimentally. Between feeding periods, insects were kept in an artificial nest under temperature and humidity closely corresponding to seasonal ones. The character of the agent transmission was estimated according to the fraction of fleas with the agent in the aggregated state (bacterial lumps, partial blocks of proventriculus), the fraction of blocked individuals, and the fraction of infected susliks and of those with the generalized form of infection. Seasonal dynamics of epizootic process of the Y. pestis transmission corresponded to the results obtained in the epizootic examination of the Tuva natural plague focus and reflected the dynamics of the epizootic process (increase-peak-decline). The activity of the formation of a proventriculus block in C. t. altaicus, the infection ability of the fleas, and the sensitivity of long-tailed Siberian susliks to Y. pestis were the highest in mid-summer (July-first ten days of August), during the period of epizooty activation in the focus. The maximal number of C. t. altaicus with the plague agent at the aggregated state was observed in the cold period, before wintering of insects and after their hibernation.     

Enhanced blocking capacity of the plague microbe in the body of the flea

During the stay of plague microbe in the organism of flea, under the effect of bactericidal factor increases its ability to form the block of proventriculus in these insects. Thus, in fleas infected on white mice, which were infected in a natural way through blocked individuals, the block of proventriculus appears 2 to 3 times as often (59.5 and 34.0%) as in insects (15.02%) infected on animals, which were infected artificially.     

Serotype differences and lack of biofilm formation characterize Yersinia pseudotuberculosis infection of the Xenopsylla cheopis flea vector of Yersinia pestis

Yersinia pestis, the agent of plague, is usually transmitted by fleas. To produce a transmissible infection, Y. pestis colonizes the flea midgut and forms a biofilm in the proventricular valve, which blocks normal blood feeding. The enteropathogen Yersinia pseudotuberculosis, from which Y. pestis recently evolved, is not transmitted by fleas. However, both Y. pestis and Y. pseudotuberculosis form biofilms that adhere to the external mouthparts and block feeding of Caenorhabditis elegans nematodes, which has been proposed as a model of Y. pestis-flea interactions. We compared the ability of Y. pestis and Y. pseudotuberculosis to infect the rat flea Xenopsylla cheopis and to produce biofilms in the flea and in vitro. Five of 18 Y. pseudotuberculosis strains, encompassing seven serotypes, including all three serotype O3 strains tested, were unable to stably colonize the flea midgut. The other strains persisted in the flea midgut for 4 weeks but did not increase in numbers, and none of the 18 strains colonized the proventriculus or produced a biofilm in the flea. Y. pseudotuberculosis strains also varied greatly in their ability to produce biofilms in vitro, but there was no correlation between biofilm phenotype in vitro or on the surface of C. elegans and the ability to colonize or block fleas. Our results support a model in which a genetic change in the Y. pseudotuberculosis progenitor of Y. pestis extended its pre-existing ex vivo biofilm-forming ability to the flea gut environment, thus enabling proventricular blockage and efficient flea-borne transmission.     

Differences in the blocking of the proventriculus in male and female Xenopsylla cheopis (Siphonaptera: Pulicidae)

Frequency of block formation was studied in Xenopsylla cheopis infected with the triptophan-dependent and the typical strains of Yersinia pestis from the Mountain-Altai natural plague focus. It was shown that the rate of blocked males in comparison with that of females was statistically higher. In experiments with the triptophan-dependent strain the frequency of blocking in males was 40.9% and exceeded that in females at four times. The highest rate of blocked males was marked at 7-10th and that of females at 21st days. The tryptophan-dependent strain did not concede to the typical strain in the ability to form a proventricular block in fleas.     

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).     

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.     

Inhibitor production, a stable feature of plague pathogen

Application of the methods developed by the author to the plague causative agent isolated in six natural plague foci provided detection of its ability to synthesize a factor inhibiting the growth of the producing strain and cultures isolated from the same focus. In the strains isolated from these foci the inhibitor presence was stable on the agar consisting of fermentative casein hydrolysate and 0.6 per cent of lithium chloride when the indicator strains were incubated under definite temperature conditions. The results of the present study and previous studies of the author, as well as the literature data suggested that the ability to synthesize the inhibitor is a stable feature of the plague causative agent.     

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfPhi), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfPhi forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfPhi is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfPhi genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfPhi genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfPhi despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.     

Epizootiological importance of Frontopsylla hetera (Siphonaptera) fleas in the Gorno-Altai natural plague focus

Experiments conducted during all seasons have established that F. hetera, one of the mass species of fleas in Mountain Altai, can be infected both by the strain of selective virulence typical to this nidus and by the non-typical non-virulent mountain-altai strain of plague agent. The non-virulent strain does not form in fleas the block of proventriculus and within 1.5-2 months they become free from the microbe. At the infection with the typical strain of the altai subspecies rare transmissions of the agent to Pallas' pika can take place as well as its long preservation in fleas.     

The influence of the host on the frequency of block formation in males and females of Citellophilus tesquorum altaicus Ioff, 1936 (Siphonaptera: Ceratophyllidae) and transmission of the plague pathogen by these fleas

Data obtained during feeding of infested females and males of Citellophilus tesquorum altaicus Ioff, 1936 (Siphonaptera: Ceratophyllidae), the main vector of plague in Tuva natural plague focus, on the natural hosts and on laboratory animals were analyzed. Sexual differences in fleas were found to depend on the host. Females fed more actively on longtailed ground squirrels Spermophilus undulatus than on white mice. The feeding activity of males on the two hosts did not differ. A higher mortality of infested females and males was observed during feeding on mice. The frequency of block formation and pathogen transmission in males was higher during bloodsucking on the ground squirrel; in females, during feeding on mice. Thus, sex-related differences in the plague pathogen transmission revealed in laboratory white mice may be not the same in nature. This should be taken into account when extrapolating the experimental data on the natural relations between the plague pathogen and ectoparasites.     

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.     

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.     

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.

     

Potential Effects of Environmental Conditions on Prairie Dog Flea Development and Implications for Sylvatic Plague Epizootics

Fleas are common ectoparasites of vertebrates worldwide and vectors of many pathogens causing disease, such as sylvatic plague in prairie dog colonies. Development of fleas is regulated by environmental conditions, especially temperature and relative humidity. Development rates are typically slower at low temperatures and faster at high temperatures, which are bounded by lower and upper thresholds where development is reduced. Prairie dogs and their associated fleas (mostly Oropsylla spp) live in burrows that moderate outside environmental conditions, remaining cooler in summer and warmer in winter. We found burrow microclimates were characterized by stable daily temperatures and high relative humidity, with temperatures increasing from spring through summer. We previously showed temperature increases corresponded with increasing off-host flea abundance. To evaluate how changes in temperature could affect future prairie dog flea development and abundance, we used development rates of O. montana (a species related to prairie dog fleas), determined how prairie dog burrow microclimates are affected by ambient weather, and combined these results to develop a predictive model. Our model predicts burrow temperatures and flea development rates will increase during the twenty-first century, potentially leading to higher flea abundance and an increased probability of plague epizootics if Y. pestis is present.

     

Epizootological role of fleas in the Gorno-Altai natural plague focus (a review)

Epizootological role of fleas in the Gorno-Altai natural plague focus (Sailugemsk focus) and numerous data on the flea viability are analyzed and generalized. Information concerning the flea natural infectivity with Yersinia pestis altaica is represented. Ecological peculiarities of some flea species parasitizing the main host, Mongolian pika Ochotona pallasi, and nature of their interrelations with Y. pestis are investigated. It is shown that the flea taxocenosis provides the permanent all year-round circulation of Y. pestis in the Gorno-Altai natural focus. Certain combinations of structural elements of the flea taxocenosis have a dominant significance in determination the circulation process at different phases of the annual epizootic cycle.     

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.     

Lysogeny by phages specific to Yersinia pestis in bacteria of the microbiocenoses of wild plague-carrying rodents

For the first time lysogenic bacteria of the genus Escherichia, spontaneously producing bacteriophages specific to the infective agent of plague, have been detected in the microbiocenoses of midday gerbils, plague carrier in the Volga-Urals natural focus. The morphological, cultural and biochemical properties of 22 lysogenic Escherichia strains isolated from wild rodents, as well as the spectra of the lytic activity of temperate bacteriophages produced by the above-mentioned microorganisms have been studied. Proceeding from the findings obtained in this research, all the strains have been divided into 3 lysotypes.