Citellophilus Tesquorum Research Articles

Bartonella, Blechomonas and Trypanosoma in fleas from the long-tailed ground squirrel (Spermophilus undulatus) in northwestern China

Fleas are known to be vectors for a variety of pathogens in veterinary medicine. However, no information is available on the presence of Bartonella and Trypanosomatidae in fleas of the long-tailed ground squirrel (LTGR, Spermophilus undulatus). The present study shows detection of these pathogens in LTGR fleas. During 2022–2023, a total of 396 fleas were collected from 91 LTGRs in 4 alpine regions of Xinjiang Uygur Autonomous Region (northwestern China) and grouped into 54 flea pools. Flea species were identified according to morphological characteristics and molecular data. In addition, all flea samples were analyzed for Bartonella with amplification and sequencing of a 380-bp part of the gltA gene and Trypanosomatidae with targeting the 18S rRNA (850-bp) and gGAPDH (820-bp) genes. The flea species included Frontopsylla elatoideselatoides (203), Neopsylla mana (49), and Citellophilus tesquorum dzetysuensis (144). Of 54 flea pools, seven (12.96%) tested positive for Bartonella, and three (5.56%) were positive for Trypanosomatidae. Based on BLASTn and phylogenetic analyses, i) Bartonella washoensis in F. elatoideselatoides and C. tesquorum dzetysuensis, and Bartonella rochalimae in F. elatoideselatoides were identified. Interestingly, a new haplotype within the species Ba. washoensis was discovered in C. tesquorum dzetysuensis; and ii) Blechomonas luni was confirmed in C. tesquorum dzetysuensis and Trypanosoma otospermophili in F. elatoideselatoides. Two Bartonella species and two Trypanosomatidae members were discovered for the first time in fleas from LTGRs. This study broadens our understanding of the geographic distribution and potential vectors for Bartonella and Trypanosomatidae.

On Microflora of Entomoparasitic Nematodes from Long-Tailed Souslik Fleas in Tuva Mountain Natural Plague Focus

Entomoparasitic nematodes have a specific role to play in the persistence of Yersinia pestis in natural foci, however their natural microbial environment has not been studied.The aim of the study was to search for bacteria associated with nematodes parasitizing fleas-vectors of plague in the Tuva natural focus.Materials and methods. Fleas were collected during the planned epizootiological surveillance in May, 2017 and 2018. During the taxonomic identification of insects, the presence of parasitic nematodes was recorded. In 2017, bacteriological examination of fleas affected by nematodes and those free from them was carried out separately without dissection. The presence and number of bacteria colonies on the agar plates were taken into account. The results were evaluated by conventional methods using the Excel program. The t-criterion, one-factor analysis of variance were employed. In 2018, 84 invaded fleas were dissected. Extracted nematodes were suspended in Hottinger’s broth, followed by cultivation of grown bacteria on Hottinger’s agar. Systematic position of isolated cultures was determined through mass spectrometric analysis (MALDI-TOF) and partial sequencing of 16S rRNA.Results and discussion. In 2017, 30 samples of fleas with helminthes and 276 without helminthes were bacteriologically examined, including 23 samples infested and 145 non-infested specimens of the main vector, Citellophilus tesquorum. Statistical differences in the proportion of samples contaminated by unpretentious bacteria and effect of flea abundance in the sample on the number of colonies grown have not been revealed. In 2018, 26 cultures of bacteria of the genera Serratia, Staphylococcus, Pseudomonas, Acinetobacter, Macrococcus, and Bacillus were isolated from 23 samples of entomoparasitic nematodes. Possible ways of their penetration into flea imagoes are discussed.

The <i>Yersinia pestis</i> aggregation as a functional adaptation to the flea organism (review)

The understanding of mechanisms and conditions of plague microbe aggregation in vectors (fleas) organisms has been of interest for a long time, and, with start of biofilm research in 1990s, it got a new perspectives.
 With the aim to determine the characteristics of the plague microbe aggregation in the organism the main vector in Tuva natural plague focus flea of Citellophilus tesquorum species, the data of many years of experimental studies focused on typical virulent strains of Yersinia pestis subsp. pestis were summarized and analyzed. Ectoparasites were infected and fed on long-tailed ground squirrel (Spermophilus undulatus), which is their natural host and main carrier of plague in Tuva natural plague focus. Interaction between Y. pestis and fleas were estimated using the rate of individuals with conglomerates bacterial lumps, formed during feeding, and blocks in the proventriculus foRmed during feeding and throughout the whole experiment registered in alive fleas after bloodsucking, and also using frequency of causative agent transmission to animals used for feeding.
 The analysis identified factors influencing the frequency and dynamics of different Y. pestis aggregate types forming into C. tesquorum, and suggest that these processes are functional adaptation of microbe to the flea organism.

The Infestation of Fleas of the Long-Tailed Souslik with Entomoparasitic Nematodes in Tuva Mountain Natural Plague Focus

Entomoparasitic nematodes are supposed to be a link between parts of Yersinia pestis population in the environment and the flea vector. The aim of the study was to assess the prevalence and intensity of infestation in the fleas of the long-tailed souslik with entomoparasitic nematodes on the territory of Mongun-Taiginsky station in the Tuva natural plague focus. Materials and methods. Fleas were collected during the scheduled epizootiological surveys in 2019–2021. In the course of taxonomic identification the presence of parasitic nematodes was registered. In order to evaluate the intensity of nematode invasion, a total of 190 fleas were dissected. The number of adult parasitizing females and presence of larvae was recorded. Statistical processing of the data was performed with the help of conventional methods using the Excel software. The criterion χ2 was applied; the influence of various factors (species, gender of fleas) on the studied parameters was assessed through single- and two-factor analysis of variance. Results and discussion. During three years of observations, entomoparasitic nematodes were found in six species of fleas: Citellophilus tesquorum, Frontopsylla elatoides, Rhadinopsylla li transbaikalica, Frontopsylla hetera, Oropsylla alaskensis, and Neopsylla mana. The differences in infestation with nematodes between the species are presented. The highest invasion rate – 25.1–25.6 % – is observed in Rh. li transbaikalica. The gender of leas does not influence their infestation. It is established that invaded fleas are more often found in the nest than in the fur of animals, they are less actively migrate to the burrow entrance compared to not invaded ones. Evaluation of infestation prevalence has revealed that fleas Rh. li transbaikalica are the hosts for nematodes of mono- or oligoxenic species, which do not occur in other fleas.

ИЗУЧЕНИЕ ЧУВСТВИТЕЛЬНОСТИ БЛОХ К ПРЕПАРАТАМ НА ОСНОВЕ ХЛОРПИРИФОСА И ЗЕТАЦИПЕРМЕТРИНА

The results of the study of the sensitivity of fleas – vectors of the plague microbe to insecticidal preparations based on chlorpyrifos and zetacypermethrin are presented. Studies were carried out on imago fleas of three laboratory cultures: Xenopsylla cheopis Rothschild, 1903 (Siphonaptera, Pulicidae), Citellophilus tesquorum Wagner, 1898 and Nosopsyllus laeviceps Wagner, 1909, (Siphonaptera, Ceratophyllidae). During the experiment, it was found that preparations based on zetacypermethrin and chlorpyrifos were highly insecticidal for Xenopsylla cheopis (S-XC), Citellophilus tesquorum (S-CT) and Nosopsyllus laeviceps (S-NL) fleas. The diagnostic concentrations found for insecticides using sensitive populations of S-shutters (S-XC, S-CT, and S-NL) were determined. The results obtained may be in demand in practice by specialists who carry out work on the regulation of flea numbers during field and village pest control in natural plague foci in the North Caucasus. Key words: fleas, insecticide, plague, natural focus.

Genetic Evidence of an Isolation Barrier between Flea Subspecies of Citellophilus tesquorum (Wagner, 1898) (Siphonaptera: Ceratophyllidae).

Simple SummaryWe studied field collections of two flea subspecies that are plague vectors on the vast Palearctic territory. Analysing the molecular–genetic, geographical, morphological, and reproductive isolation criteria, we conclude that these subspecies could be considered different species.This study investigated the relationship between two subspecies of the Citellophilus tesquorum flea, C. t. altaicus and C. t. sungaris, which are vectors of the bacterium Yersinia pestis that causes human plague across the vast territories of the Palearctic. Adult fleas were collected from 16 localities and 11 populations in 2019 and 2020. Specimens were morphologically verified for subspecies status and analysed for mitochondrial cytochrome c oxidase subunit I (COI) DNA, nuclear ribosomal cluster internal transcribed spacer 1 (ITS1) and ITS2, and Wolbachia-infection status. Our results demonstrated a genetic difference between C. t. altaicus and C. t. sungaris. According to mitochondrial data, the genetic distance between clades of C. t. altaicus and C. t. sungaris was comparable with the species divergence of the genus Callopsylla, which is closely related to Citellophilus. All studied populations of C. t. altaicus were Wolbachia-infected, whereas all studied populations of C. t. sungaris were symbiont-free. Data for ITS1 and ITS2 had much lower phylogenetic signals than mitochondrial data; however, diagnostic substitutions for C. t. altaicus and C. t. sungaris delimitation were also revealed. Analysis of a hardly accessible report on cross experiments allowed us to conclude the partial postzygotic isolation between these subspecies. Taken together, the molecular-genetic, geographical, morphological, and reproductive isolation findings verified that C. t. altaicus and C. t. sungaris subspecies could be considered as different species.

Diversity of Fleas (Siphonaptera), Vectors of Plague Pathogens: the Flea Citellophilus tesquorum (Wagner, 1898), a Parasite of Ground Squirrels of the Genus Spermophilus

This paper presents the results of analysis of the taxonomic diversity, distribution, and host associations of the flea species recorded so far as the plague microbe vectors. The ecological features and epizootic significance of the flea Citellophilus tesquorum (Wagner, 1898), a parasite of ground squirrels, are specially considered.

The Effect of Initial Infection of Citellophilus tesquorum altaicus Ioff, 1936 (Siphonaptera: Ceratophyllidae) with Yersinia pestis on Its Alimentary Activity, Mortality, and Biofilm Formation

Alimentary activity and mortality was assessed in fleas Citellophilus tesquorum altaicus non-infected with Yersinia pestis and those with initial infection levels 50 and 100% during feeding on a non-specific host (white mice). The presence of the plague pathogen in fleas significantly stimulated their feeding activity, especially in females. No effect of infection on flea mortality was observed. At the same time, male fleas died more frequently than females.

Relationship of the Representatives of Eight Genera of Siphonaptera Order and Yersinia Pestis from Tuva Natural Plague Focus

Mutual relations of Yersinia pestis and fleas are a necessary condition to estimation the role of carriers in maintenance of the causative agent circulation. Objective – the comparative analysis of frequency of «conglomerate» and «block» formations by Y. pestis in fleas parasitizing on the main and minor carriers in the Tuva natural focus. Materials and methods. Results of experiments with representatives of eight genera of Siphonaptera order and Y. pestis subsp. pestis from the Tuva focus were analyzed. Mammals with various epizootological roles in the focus were used as flea fooders. Frequency of «block» and bacterial «conglomerate» formations and death of the infected insects was estimated. Results and discussion. The highest level of block formation was observed in Citellophilus tesquorum altaicus, Frontopsylla elatoides, Neopsylla mana, Oropsylla alaskensis, Rhadinopsylla li transbaicalica – parasites of the basic carrier (Spermophilus undulates). Formation of «conglomerates» was more often revealed in F. hetera, Paradoxopsyllus scalonae, P. scorodumovi, P. dashidorzhii, Amphipsylla primaris – ectoparasites of the minor carriers (Ochotona pallasi, Alticola strelzovi). Positive connection between frequency of Y. pestis conglomerate formations in fleas and their death rate was detected. Maximal death rate of the carrier was observed at high degree of Y. pestis aggregation. It was supposed that fleas of the basic carrier participated in a greater degree in Y. pestis distribution and ectoparasites of the secondary carriers – in its persistence.

INFLUENCE OF YERSINIA PESTIS PLASMID CONTENT ON BIOFILM FORMATION IN FLEAS WITH DIFFERENT VECTOR ACTIVITY

Aim. Influence of the plague agent plasmid content on biofilm formation in vivo and death rate of fleas-vectors with different vector activity in experiment were analyzed. Materials and methods. Three Yersinia pestis strains: virulent I-3230 (pYT, pYV, pYP) and I-2638 (pYT, pYV, pYP, pTP 33), and its selected avirulent isogenic clone I-3480 lacking two plasmids (pYV, pYP) were used. Three species of fleas were artificially infected: 477 individuals of Xenopsylla cheopis (a highly active vector), 441 - Citellophilus tesquorum (an active vector), 519 - Frontopsylla lucu-lenta (a low-active vector). The peculiarities of Y. pestis biofilm formation in fleas were estimated by a portion of individuals with bacterial «conglomerates» and «blocks» for a feeding. Death rate of the insects was defined by the percent of the dead fleas at each feeding. Results. All three flea species infected by Y. pestis strains carrying an additional plasmid pTP33 (I-2638 and I-3480) demonstrated the increase of the individual number with various biofilm forms in comparison with the three-plasmid strain I-3230. In X. cheopis it occurred due to the blocked insects, in C. tesquo-rum - mainly due to the fleas containing «conglomerates», in F. luculenta it was completely connected with ectoparasites with «conglomerates». A share of X. cheopis and C. tesquorum died at a feeding was higher in ectoparasites infected with I-3230 strain and F. luculenta - infected by I-2638. Conclusion. Y. pestis strains possessing an additional replicon pTP33 formed a biofilm in the infected insects more often and larger size than a classical three-plasmid variant. Influence of the strain plasmid content on death rate of the infected fleas depended on a vector species.

PECULIARITIES OF INTERACTION BETWEEN CITELLOPHILUS TESQUORUM ALTAICUS (IOFF, 1936) FLEA AND YERSINIA PESTIS WITH VARIOUS PLASMID COMPOSITION

Objective of the study is in vitro investigation of mutual relations between Citellophilus tesquorum altaicus and Yersinia pestis with various plasmid composition: influence of the strain on flea alimentary activity and mortality rate, frequency and dynamics of biofilm formation. Materials and methods . C. tesquorum altaicus were infected by three Yersinia pestis strains: virulent triple-plasmid I-3230 isolated in Mongolia, referential for the Tuva focus I-2638 carrying four plasmids (pYT, pYV, pYP, pTP 33) and also selected from it avirulent isogenic clone I-3480 that lost two plasmids (pYV, pYP). Peculiarities of interaction between fleas and Y. pestis strains were estimated through the lens of specimens with «conglomerates» and “blocks” for feeding, the period from infection prior to the beginning of conglomerates’ formation, alimentary activity, and mortality rate of the infected fleas. Results and conclusions . It was revealed that alimentary activity of the infected insects was higher than that of the control group, and the highest – in fleas infected with I-2638 strain. Greater numbers of dead fleas at feeding was noted in specimens inoculated with I-3230 strain. Predominant significance of I-2638 strain was established in C. tesquorum altaicus biofilm formation both as «conglomerates» and “blocks”. I-3480 strain also formed the conglomerates in fleas more actively than I-3230 lacking pTP33 plasmid. Thus, four-plasmid I-2638 strain surpassed triple-plasmid I-3230 and two-plasmid I-3480 strains in reference to all tested indicators except flea mortality rates. It may testify to co-adaptation of Y. pestis and C. tesquorum altaicus from the Tuva plague focus and to the possibility of a pTP33 functional role in enhancement of a biofilm formation in vivo.

Characteristics of Entomoparasitic Nematodes of Small Rodent Fleas

Entomoparasitic nematodes are the natural regulators of numbers of fleas – plague vectors in natural plague foci. They have an impact on the intensity of epizootic process, progressing in these foci. Objective of the study is to examine entomoparasitic nematodes from fleas of the souslik and vole, collected in the territory of Povolzhsky Region. Materials and methods. Presented are the results of morphological and genetic analysis of entomoparasitic nematodes, flea parasites – Citellophilus tesquorum, Amphipsylla rossica and Ctenophthalmus secundus, collected from the small ground squirrel Spermophilus pygmaeus and social vole Microtus socialis, respectively. Results and conclusions. Established has been morphological identity of nematode parasitic females isolated from all the three species of fleas, and a high degree of similarity to the previously described Rubzovinema ceratophylla, parasitizing on C. tesquorum fleas. Substantiated has been the belonging of parasitic nematodes isolated from fleas C. tesquorum, А. rossica and C. secundus, to one and the same species, specified as Rubzovinema sp., based on the high percentage of the homology of the nucleotide sequences of rRNA genes (99.3–100 %). This species differs from the previously described monoxenous R. ceratophylla in absence of strict host specificity.

Epizootiological Consequences of the Possible Importation of Plague Agent from Manchuria in the Territory of Trans-Baikal Natural Focus

Experimental investigations have demonstrated that proventriculus of Citellophilus tesquorum sungaris (population habitant in the territory of Trans-Baikal region) and Xenopsylla cheopis (classical vector) is blocked in case of the infestation with the plague microbe strain isolated from humans in Manchuria (China) in 5,6 and 15,4 % of the instances, respectively. These ectoparasites transmit plague agent to white mice both when infected with plague microbe at the moment of clustered/group feeding, and when they have their proventriculus blocked at the moment of individual feeding. Taking into consideration ecology of the species of fleas under discussion and sensitivity of the local rodent population to plague microbe, one cannot exclude possibility of the local epizooty occurrence in the territory of Siberian and Far East regions in case of plague agent importation from China.

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 Citellophilus tesquorum aitaicus Ioff, 1936 infested females and males (Siphonaptera: Ceratophyllidae), the main vectors of plague in Tuva natural plague locus, on the natural host and laboratory animal was analyzed. It was found that sexual differences in fleas depended on the type of the host. Females fed more actively on the longtailed ground Citellus undulatus than on white mouse. Alimentary activity of males on these animals was similar. Higher mortality of fed females and males was noted during feeding on mice. Frequency of formation of the "block" and transmission of the pathogen in males was higher during bloodsucking on the ground squirrel; in females, during feeding on mice. Thus, differences in the transmission of the plague pathogen, revealed in laboratory on white mice, can be quite different in nature. So, extrapolation of experimental data on natural processes of interrelations between plague pathogen and ectoparasites must be performed taking into account revealed peculiarities.

Discussion on preservation mechanism of Yersinia pestis by Citellus undulatus

AbstractStudies prove that plague in Citellus undulatus exists in the nature with the pattern of successive prevalence every year. Citellophilus tesquorum altaicus is not only the main transmission vector, but also the reservoir of C. undulatus plague. And the latter can spread Yersinia pestis. The fact challenges to the traditional theory of natural focus and carries out a theory breakthrough.

Seasonal dynamics of epizootic process of the plague agent Yersinia pestis transmission to the long-tailed suslik Citellus undulatus by the flea Citellophilus tesquorum 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.