A.N. ALEKSEEV, H.V. DUBININA
EditorinChief - A.F. Alimov - Director of the Zoological Institute RAS
V.V. Khlebovich, Ya.I. Starobogatov, S.D. Grebel’nyi,
T.A. Asanovich, Yu.S. Balashov, V.Ja. Berger, I.S. Darevsky,
V.R. Dolnik, S.Yu. Kuznetsov, À.V. Gorokhov
Editor of the volume:
T.K. Sergeeva, I.O. Alyakrinskaya
The bases of parasite—vector system stability were analyzed. For this purpose
as examples were used plague, malaria and tick-borne infections. Anthropogenic
activity consequences on the natural foci of diseases were analyzed. It was
stressed that this activity made the situation worse promoting sometimes the
circulus viciosus (changed environment—vector—pathogen—man) appearance. An
attempt was made to analyze the interrelationships between different pathogens
in the cases of vector multi infections and to estimate such phenomena for man.
This publication is financed by the Russian Foundation for Basic Research (grants No. 96-04-48531 and No. 02-04-48654).
This series is proposed to publications of separate works from the Zoological Institute in Russian and English. It is published without any periodicity and distributed according to request applications.
© Zoological Institute RAS, 2002
© A.N. Alekseev, H.V. Dubinina, 2002
Zoological Institute RAS, 199034, St. Petersburg, Universitetskaya nab., 1
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In the number and diversity of species inhabiting the Earth parasites exceed free-living forms, by at least one order of magnitude. They play a great role in the circulation of matter and energy, exchange of genetic material between themselves and their hosts and exchange between hosts, and therefore in the evolution of diverse living forms on the Earth. Their role as markers of the state of external environment, monitoring of which becomes increasingly important for the survival of species Homo sapiens is difficult to overestimate. The chains of cause and effect modified by man cannot but affect the parasitic constituent of the biota and return to man like a boomerang having an impact on the state of its populations.
The consideration of separate short chains of cause and effect is characteristic of foreign science: the aim of such discoveries is often to clarify the role of one factor, which is frequently quite important, associated with a specific parasite, and very seldom it includes analysis of functioning of the system within a biocenosis and all the more so within the biota. The importance of works of Bacot and Martin (1914) who proved the role of synanthropic rats in the transfer of plague is invaluable. However long before that D.K. Zabolotnyi (1899) noticed that outbreaks of plague may be associated with the natural system having no anthropogenic changes where epizootia of plague are maintained in populations of Siberian marmots. Further the theory of Academician Pavlovsky on the natural focality of a number of transmissive diseases developed and supplemented by his disciples provided profound theoretical corroboration of plague endemicity. Russian works show that plague natural foci have been in existence for many millions of years over extensive areas the Earths surface, including millions of square kilometres nearly untouched by human activity. These works revealed relationship between functioning of endemic and synanthropic foci of this disease. In particular, Russian scientists conducted a profound study of the mechanisms providing for stability of this system. It now includes not only telluric hypothesis a possibility of preservation of Yersenia pestis in the semisaprophyte state in the soil, but also a possibility of regulation of the pathogen owing to feeding of fleas on argasid ticks containing huge amounts of plague microbes. We permitted ourselves to generalise this pattern as mosaic of works of many Russian authors in the form of one scheme reflecting our ideas (Alekseev, 1996b) on the ways of circulation of this microbe, which provide for its distribution and stability of its preservation in time and space (Fig. 1).
Fig. 1. Transmision and circulation routes of .plague agent
I – system: flea, Yersenia pestis and susceptible rodent; II – generalized and aerial forms of plague; III – Ornithodoros as a “living can” of Y. pestis for flea; IV – possible phytophase of the Y. pestis cycle (telluric hypothesis); 1 – Y. persis in the rodent blood; 2 – blood and agent consumption by the susceptible flea; 3 – agent development, reproduction and transformation in the flea vector; 4 – transmission of agent by blocked hungry flea; 5 – transmission of agent to man, plague bubo formation and generalisation of infection; 6 – pulmonary form of plague; 7 – transmission by coughing to a healthy person.
It took 20 years after the discovery of movable microgametes in a drop of blood of to infect the vector mosquito of the genus Anopheles. That was done by Ronald Ross, a British doctor, researcher, writer and mathematician, who was the first to considering pathogen and its vector not only as a certain system (in 1898 he traced for the first time the complete cycle of development of bird malaria pathogen). He was also the first to pay attention to the role of vectors habitat in particular to the fact that human activity can increase vectors abundance. This outstanding researcher has noted for the first time the role of small fish in water bodies as biological enemies of larvae of malaria mosquitoes and substantiated the possibility of mathematical calculation of the minimum of vectors when the transmission can be interrupted. (Ross, 1911) The study of Ross is the exception among the foreign parasitological works on the turn of the century. All these data were obtained in India where in a century malaria is still one of the most widespread diseases.
The bases of ecological approach to the impact on the parasitic system, among ones having the most simple structure where man is the donor and recipient and the vectors are mosquitoes of one genus Anopheles were therefore present. However only Russian researchers such as E.I. Martzinovsky (Rashina, 1954) and following him V.N. Beklemishev the author of the most comprehensive monograph for that time entitled Ecology of malarial mosquito (1944) could appreciate and use to full extent the possibility of an ecological approach to the destruction of the basis of malaria pathogen circulation in human populations. They justly assumed that it would be impossible to maintain permanently vector numbers on the level below the critical one for transmission, and they choose strategy of impact on donors of pathogen (treatment) and on recipients (chemoprophylaxis) and vector control. As a result malaria as a mass disease was exterminated in the former Soviet Union in the 1950s. Anophelism without malaria posed (and still poses) potential danger on all primarily endemic territories. The system may begin functioning again at any moment, since its basis i.e., sensitive vector cannot be eliminated from nature. An attempt of the World Health Organisation to eliminate malaria by means of eliminating vector lead only to appearance of Anopheles race resistant to most diverse insecticides; mankind is so far unable to affect to a sufficient extent all the three links of the system, which appeared to be the most simple anthroponosis malaria.
Separate attempts to suppress malaria by means of medical drugs and chemophrophylaxis led to gradually increasing spread out of pathogen forms resistant to medical treatment. The system changed by many, e.g. rearing of DDT resistant vector races changes the character of its functioning. We have shown that manifold increase of DDT resistance in Anopheles sacharovi in Azerbaijan changes the nature of the epidemiological process shifting it in season and increasing the number of late relapses of three day-malaria. Malaria in Azerbaijan acquired in the 1970s northern nature, probably as a result of the fact that resistance to cold and DDT is provided by a similar group of genes in genome of Anopheles (Alekseev, Baranova, 1985).
It was typical of V.N. Beklemishev to have not only a comprehensive ecological approach to parasitic diseases of man, but biocenological, evolutionary one. The posthumously published collection of his works Biocenological bases of comparative parasitology (1970) continue being a source of fruitful ideas.
V.N. Beklemishev (1948) for the first time in the world science tried to compare systematic position of vectors, belonging to very different orders of blood-sucking invertebrates, and their possibilities to transmit to terrestrial vertebrates all known pathogens of human and animal diseases. His reasoning was based upon analysis of evolutionary ancient age of vectors (mostly blood-sucking) feeding on vertebrates and their possible contacts with agents pathogenic to their hosts. These ideas were transformed into a matrix, groups of vectors (from an order to genus) plotted along the vertical axis, and large groups of pathogens, which they can or cannot transmit being their invertebrate hosts and being at leas temporary sources of feeding of these pathogens.
In the late 1940s when this scheme was created far not all pairs vector-pathogen were studied and classified correctly. The cursory characterisation given for the notion specificity as the presence of pathogen in host (Beklemishev, 1948) contained no sufficient and reasonable answer to the very important question put by E.N. Pavlovsky (1947) a year before the paper by V.N. Beklemishev appeared: why far not all bloodsuckers having an opportunity to receive most diverse pathogens become their vectors. The answer to this question is the key to understanding stability of parasitic systems. Evolutionary relationships deciphered by V.N. Beklemishev provided explanation of their appearance in the past, restricting the possibility of predict prohibitions for discovery of stable or ephemeral systems.
V.N. Beklemishev analysed only possible phylogenetic relationships of pathogens and bloodsuckers and only briefly touched upon questions of what peculiar features of ontogenesis, morphology, physiology and behaviour determine possibility or impossibility of formation of representatives of one or other systematic group of arthropods by specific vectors of strictly determined pathogens of diseases of terrestrial vertebrates.
Therefore the question posed by E.N. Pavlovsky as early as 1947 had never been considered by anyone, and we tried to apply this to specific pathogen vectors (excluding mechanical ones). To answer this question we had to define what we understand as specific vectors among which we included specific contaminators. Without going into detailed substantiation of the proposed criteria, as this was done in a special review (Alekseev, 1984), we will list these criteria:
Fulfilment of the first and the second requirements of specificity depend not only on the systematic position of vectors, but also on the character of their metamorphosis, types of feeding of preimago and imago. Beklemishev (1948) briefly mentions this circumstance. Working on fleas, highly efficient vectors of plague and highly virulent strains of pathogen released from them, infecting vectors in doses (Alekseev et al. 1967) we had a possibility to become convinced that a large portion of microbes dies under the action of bactericidal factors of vector intestine (Alekseev, et al. 1969, 1972; Bibikova, Alekseev, 1969) and only a part of the population transforming (Bibikova, Klassovsky, 1974) and reproducing can infect vertebrate host (Bibikova et al., 1968). Larvae of fleas are not only detritophages, but also blood-eaters (Strenger, 1973); bactericidal factors of Aphanipetera only partly render bacteria harmless. Lysozymes of ticks, to judge by the data of V.M. Podboronov and A. Berdyev (1991), render bacteria harmless completely or partly the majority of Bacteriaceae infecting them (Yerseniae and Francisellae are an exception), however they can favour transmission of specific and apparently the most ancient symbiont virus of tick-borne encephalitis (Alekseev et al., 1995b, c).
Considerations on the role of ontogenesis and types of feeding of preimago and imago of specific vectors permitted us to formulate the theory of relationships of types of feeding and digestion of bloodsucking arthropods and their ability to be specific vectors of agents of transmissive diseases (Alekseev, 1985) and hence prohibitions to transfer definite pathogens by definite groups of bloodsucking (or at least blood feeding) invertebrates.
The prohibitions are as follows:
We have not revealed factors restricting transmission of filariae.
Only one example of specific transfer of Trypanosoma rangeli Tejera (with saliva) and reproduction with highly efficient release with faeces during feeding of Trypanosoma cruzi Chagas is known so far for insects with double glycocalyx. Apparently only trypanosomes, including also T. cruzi (in the body cavity of bug Dipetalogaster maximus) and some viruses (Fort-Morgan in Cimicidae, Rush et al., 1980) can overcome protection barrier of bugs intestine. In the intestine cavity of Triatominae, as is known nearly any microorganisms can easily reproduce, whereas specific transmission except trypanosomes has not been established.
Prohibition factors, used in the theory are apparently not quite complete. A detailed study on biochemical level to which they lead is possibly needed. However for directed estimation and search of specific vectors among insufficiently known groups of bloodsuckers our theory, as we hope, may be useful.
Even more useful, but not used sufficiently is the idea of prohibition of search of transfer of pathogens among prohibited vectors. For Russian science this is a bygone stage. A long time has passed since the experiments on search of vectors tick-borne encephalitis (TBE) virus among arthropod groups other than ixodid ticks. However ideas of flying pins, which as it appears have to be forgotten after exact identification of the notion of specific vector (Beklemishev, 1948; Alekseev, 1984), are revived from time to time. For instance, as a result of scientific boom around the discovery of spirochetes - Lyme disease pathogen - transmitted by ixodid ticks a publication appeared about a possible role of horseflies and other blood-sucking Diptera in the transfer of this pathogen. Authors of these investigations are parasitologists, fairly well known abroad (Magnarelli et al., 1987; Doby et al., 1991). Unfortunately these wrong concepts become included into methodical materials of practitioners (Rakhmanova et al., 1995).
The possibility of transfer of spirochetes by ixodid was predicted not only by our theory, but also by matrix of V.N. Beklemishev long before that. In particular, we were able to show quite recently that circulation of borreliae - pathogens of tick spirochetoses in ixodid tick populations is in principle not different from that of argasids: exchange by pathogens occurs both by sexual and omovampiric way (Alekseev, Dubinina, 1996a).
Stability of the system tick-pathogen in this case is confirmed also by the fact that not only virus as we showed before (Alekseev, Chunikhin, 1990a), but also borreliae, can be stored in the skin of recipient in the cement cone formed saliva of the tick Ixodes persulcatus (Alekseev et al., 1995a, 1996a).
The ancient age and stability of relationships of the pathogen with specific vector is confirmed, in particular by the fact that TBE virus, being in all probability of tick origin (Chunikhin, Leonova, 1985), although it is not its permanent symbiont (Beklemishev, 1959), is so firmly tied up with the vector that even the powerful, purely anthropogenic impact, such as pesticides, do no affect the ability of pathogen transmission. Our experiments with the system poison ivermectin, in which infected TBE ixodid nymphs received a sublethal dose of poison feeding on animal led to a decrease of survival ability of poisoned individuals, to an abrupt decline of their sizes and weight, but did not affect the ability of individuals that did survive to transmit TBE virus in case of a bite (Alekseev et al., 1992).
The experiments conducted at present (Alekseev et al., 1997), with sublethal doses of phosphorus organic dursban insecticide showed that presence of TBE virus in the specific vector Ixodes persulcatus Schulze (Ixodidae, Ixodinae) treated with poison somewhat exceeds survival ability of infected taiga tick nymphs, whereas Amblyomma hebraeum Koch (Ixodidae, Amblyomminae), sensitive to virus, but host exotic for its, is sharply suppressed (Fig. 2).
Fig. 2. Insecticide (dursban) action on Ixodid nymphs infected by tick-borne encephalitis virus.
1 – specific vector of TBE virus – Ixodes persulcatus; 2 – accidental, but susceptible to TBE virus host Amblyomma hebraeum.
Facts described emphasise importance of distinct determination of specificity of relations of the pathogen and vector for the assessment not only of possibilities of a successful transmission of the pathogen, but also perspectives of action on these systems and forecasting of consequences of these impacts in the environment being changed by man. One should imagine clearly that even rather strong changes in the environment in all probability will not be able to break the durable chains, providing for functioning of ancient mechanisms that are well-balanced and have many mechanisms providing for homeostasis, not all of which have been studied sufficiently up to now.
Our foreign colleagues have only started to realise, although not fully, the principle of V.N. Beklemishev (1945) about self-sufficiency and protective properties of triple parasitic systems. These principles were formulated by him for such evolutionarily formed community as the system - trypanosome - tsetse fly - antelope.
For instance there are publications on the possibility to suppress foci of Lyme disease in the USA by means of reduction of livestock of the major hosts of adult ticks - deer, or by suppression of abundance of rodent hosts of preimago. These attempts prove to be inadequate. Stability of tick infection focus is provided by a multitude and diversity of topic, phoric and trophic relations of the primary host and reservoir of tick-borne infection pathogen - Ixodes tick. Brilliant analysis of this phenomenon was made by V.N. Beklemishev in 1959 with reference to taiga tick and TBE virus. The only example of possibility of real suppression of TBE focus over extensive areas was demonstrated in Kemerovo region in Siberia where the vector was eliminated completely over thousands of hectares owing to DDT application. It hardly would be worthwhile to follow this example, because in any case such anthropogenic impact on millions of square kilometres of natural foci is scarcely possible and is hardly reasonable.
It is owing to the multitude of phoric and topic relations that foci were restored with the restoration of the critical level of abundance l of the primary vectors.
It appears reasonable to dwell on those aspects of stability of parasitic systems that are provided by the multitude of the ways of circulation of pathogen, particularly on those aspects of this issue, which were unknown when it was actively examined by E.N. Pavlovsky and V.N Beklemishev. We will dwell on the functioning of the system, beginning from its lowest level - tick-pathogen, the pathogen being intracellular parasite, RNA virus of the family Togaviridae: system taiga tickTBE virus. It is the tick encephalitis as infection capable of existing for an indefinitely long time without inclusion into it, even in the form of a blind alley, i.e. man, and unlike plague having no mechanism of transmission from man to man, served as a model for the elaboration of the theory of natural focality of diseases. This theory permits us to include the name of E.N. Pavlovsky among the names of great naturalists. The theory has determined for decades ahead the direction of study and interpretation of their results in the field of study of parasitic infections and primarily infections transmitted by bloodsucking arthropods.
The theory of E.N. Pavlovsky on natural focality of diseases (1939a) and his principle about landscape epidemiology published in Russia began to be taken into account abroad only after they were translated into English in 1966. It was not until the 1990s, that they were used by foreign epidemiologists, in particular in connection with remote-sensing methods of the study of landscapes and their seasonal dynamics for forecasting of changes in the epidemiological situation by the diseases transmitted by mosquitoes (Roberts, Rodriguez, 1994). Meanwhile the theory of landscape epidemiology was developed in the excellent study of T.A. Vorontzova(1990) who showed that the epidemical manifestation of natural foci of tick-borne infections (with reference to TBE and rickettsiosis) is the result of the centuries long interaction between pathogen ecosystems with the system of land use characteristic of a certain historic social-economic system of a society. Moreover, a period equal to approximately 3-4 decades was determined, during which the functioning of parasitocoenoses of obligatory transmissive infections changes in such a way that mass transition of pathogens from natural foci to people in ecologically optimal parts of distribution ranges becomes possible. Instead of sporadic incidence, typical of intact natural foci, mass incidence occurs. In Russia at the turn of the century factors responsible for tick-borne infection mass incidence were changes in the deforestation (continuous forest massive disappeared) and the cessation of fire treatment of land and pastures as the old restrictions of cattle pasturing were discarded and people began using forest pastures. All principles based by the authors on the analysis of the situation with TBE can be referred to a full extent to increasingly improved diagnosis of tick borellioses over the territory of Russia.
It is remarkable that opposite changes reforestation (restoring of forests in the USA) simultaneously with the wide spreading of deer, endemic of the islands off the coast of New England, on the territory of eastern and north-eastern states in North America led to thriving of the main vector of tick-borne borreliosis, i.e. Ixodes scapularis Say, in those areas, and to epidemiological manifestations of natural foci of North American tick borrelioses, to emergence of Lyme disease named after the small town where an outbreak of this infection was recorded for the first time.
The stable natural system, which provided only isolated manifestations of the disease before changes in ecological conditions of existence of vector populations, passed over to a new level - the level of epidemiological manifestation. Other tick infections spread in tick populations in the USA, i.e. babesioses, erlichioses and virus from the group of tick-borne encephalitis, are probably on this way (Johnson et al., 1996; Telford III, 1996b; Fish et al., 1996). The isolation of tick encephalitis virus (Telford III, 1996a) from ixodids ricinus group (according to the classification used by Piesman, 1989) or persulcatus group (according to an equally arbitrary definition of N.A. Filippova, 1990) is not surprising. Related groups of ixodids can have related groups of pathogens. Their thriving and successful circulation in nature and epidemiological manifestation, depend upon interaction of pathogens in the vector. However we will consider the question of systemic interaction of several pathogens in a specific vector below.
Returning to the conditions of stability of existence of parasitic systems we should emphasise that it was Pavlovsky who created the principal scheme of TBE virus in nature, which later was included into his classical manual of 1948. However he and his successors who used test animals perceptible to the virus overstated the role of vertebrate virus carriers in maintaining stability of the foci. N. Beklemishev (1959) was the first who doubted competence of this principle; in the subsequent years a number of authors (Chunikhin, Leonova, 1985; Naumov et al., 1983, 1984) proved that the focus couldnt exist owing to infection of ticks on animals with superthreshold virusemia. A hypothesis was proposed for a reverse (mediator) infection of nymphs from imago with a simultaneous feeding on large mammals (Korenber, Kovalevskii, 1977). However apart from goats none of them has virusemia close to the superthreshold one (Naumov et al., 1983). Moreover, the most simple mathematical model of retaining of the virus in transovarial and transphasic transmissions created by S.P. Rasnitsyn (1976) and based upon the data on efficiency and of TBE virus losses from phase to phase predicted a complete loss of virus with a transition from a parental to daughter generation without the sexual exchange by virus and other mechanisms of replenishing of the virus known losses. The system not only seemed to be unstable, but it also seemed to be unable to function. However it existed and it was possible to eliminate it only by eliminating the main vector completely. New facts and new understanding of the mechanisms of its functioning were needed.
The first indirect arguments supporting the concept of exchange of pathogen among adult ticks during their feeding from the common inflammatory focus were proposed by B.R. Galimov and co-authors (1989). Concurrently with these authors and separately from them we showed that in case of obviously close attachment of ticks on an animal where viremia does not arise not only exchange of virus occurs. The pathogen obtained can be transferred to the descendants by a female of a specific vector - tick I. persulcatus: the virus was discovered in the egg batch of originally uninfected female, which sucked blood near the infected one (Alekseev, Chunikhin, 1990b, c). We called this kind of exchange transsalival (Fig. 3). This route permits exchange of strains and species of virus, avirulent for animals. In particular, such data were obtained in co-operation with our Slovakian colleague with reference to low virulent strain of TBE and Langat virus (Alekseev, Weisman, unpublished data).
Fig. 3. Scheme of TBEV exchange between Ixodes persulcatus females, which are feeding near each other (transsalival virus transmission).
1 – donor of virus; 2 – recepient; 3 – focus of inflammation.
The next step was to prove that exchange of virus can occur on animal and in case of separate feeding (Alekseev, Chunikhin, 1991). We termed this distant way of virus transmission. For the first time a possibility of such exchange was shown by our British colleagues with reference to Thogoto virus and South African tick Rhipicephalus appendiculatus Neumann (Jones et al., 1987). We have also shown that ixodid saliva possesses characteristics of adjuvant-enhancer of arbovirus transmission (Jones et al., 1989; Alekseev et al., 1991b). Later our data were confirmed by foreign scientists (Labuda et al., 1991) who showed the possibility of a reverse course of the virus - from imago to nymphs and from nymphs to larvae in case of feeding on aviremic animal.
Based upon our ideas on specificity of vector, in particular upon the proposition of highly efficient way of transmission we can suppose that the distant transmission is possible even in case of removal of an attached infected tick by animal host. The intact uninfected individuals can be infected because of preservation of virus in depot in cement plug on hosts skin where the quantity of virus (and borreliae, as we showed recently) can be equal or even higher than in the entire ticks body (Alekseev, Chunikhin, 1990a; Alekseev et al, 1995a, 1996a). Our ideas about the stability of the system providing for the conservation of pathogen populations in their specific vector are based upon: 1) the role of males in the transmission of virus of TBE revealed by us (Alekseev, 1991) and its confirmation by epidemiological practice (Penjevskaja, 1989); 2) establishment of many fold increase of moving activity of individuals infected with TBE virus (Alekseev et al., 1988a; Alekseev, Dubinina, 1994) and their response to smells (Alekseev et al., 1991a); and 3) revealing of 10 times higher number of infected ticks on man than on vegetation (Melnikova et al., 1996). It is necessary to emphasize also that unlike our foreign colleagues, we have shown that distant exchange of virus is quite different between representatives of two subfamilies: Ixodinae, where there are primary specific vectors of TBE virus I. persulcatus and Ixodes ricinus (L.), and Amblyomminae among which, there are only facultative or occasional vectors or reservoirs of this virus. Ixodinae appeared to be many times more efficient donors and recipients of virus in experiments in distant transmission on the aviremic animal (Alekseev, Chunikhin, 1992).
The study of peculiarities of virus circulation in populations of taiga ticks permitted us to formulate a number of principles on those phenomena, which provide a relatively high stability of this tick-borne encephalitis system in the external environment.
The basis of stability of this system, apart from the diversity of strains of pathogen and peculiarities of infected ticks behaviour (Alekseev, 1996a), is the diversity of the ways of virus circulation. Each of these ways may have a low probability of success, but in totality they become, like threads, interwoven into a rope firmly binding the beginning and the end of the system, reproduction of virus circulation from one ab ovo to another (Fig. 4). In our scheme we tried to trace ways known at present, which are used by virus population for its reproduction in time and space, at the same time illustrating the ephemeral nature of each separate thread by known literature data.
Fig. 4. Tick-borne encephalitis virus routes of transmission.
I – “classical”(transmissive) route from egg to egg by feeding on the susceptible animals with threshold quantity of virus in the blood; Ia – man infection by virus containing goat or cow milk; II – transphasic transmission: naïve larva gets virus on infected susceptible animal; III – sexual transmission: infected male transmits virus to naïve female during copulation; IV – virus exchange between infected nymphs and naïve larvae cofeeding not near each other on the animal without viremia (distant transmission); V – virus exchange between specimens cofeeding near each other on aviremic (not susceptible to the virus) animal (transsalival transmission); VI cannibalistic route of transmission: infected male consumes hemolymph from naïve female and injected virus infected saliva in her body; 1 – naïve tick and animal: o – ova, L – larva, Lf – fed larva, N – nymph, Nf – fed nymph, ♀ – female, ♂ – male; 2 – same infected tick and animal.
I - the classical way of transovarial and further transphasic transmission (Fig. 4, I) for the first time described as early as 1940 (Pavlovsky, Soloviev, 1940). Only part of eggs in the egg batch is infected (Ilienko et al., 1970) and only in case when female parent itself had a high virus dose in the body (Kondrashova, 1975). Virus can partly be lost during transition from phase to phase (Benda, 1958) not to mention, that far not all preimago reach the next phase irrespective of presence or absence of pathogen. The very r-strategy of tick reproduction seems to postulate this. One should not forget also the nontransmissive food way of infection with milk of domestic animals (Fig. 4, Ia).
II - Highly improbable transmissive routes (Fig. 4, II) of preimago infection on an animal with a short superthreshold viremia (Naumov et al., 1983, 1984; Chunikhin, Leonova, 1985), but one of the most important ones for supporting properties of virus that are pathogenic for vertebrates. Its passage through animal amplifiers is one of the bases of durability of metaxenosis mechanisms (Beklemishev, 1959) and epidemic manifestations of the disease.
III - sexual transmission (Fig. 4, III) from males to females (Chunikhin, et al., 1983), although it can provide for transovarial transmission in only 10% of cases of copulations of infected I. persulcatus males to females, it is still provides notablesupport to the transition of virus to the following generation of ticks. It would be timely to note that the mathematical model of transphasic transmission of virus (Rasnitsyn, 1976) long before establishing the fact of sexual trasmission provided for the probability of such kind of virus transmission.
IV - distant exchange (Fig. 4, IV) by virus in case of joint feeding on aviremic animal and V - transsalival exchange (Fig. 4, V) under close joint feeding were considered by us above and are apparently some of the main ways of reverse route of the pathogen from an older phase to the younger one: from imago to nymphs and from nymphs to larvae and also exchange of virus between sister individuals of the same generation. The mechanism of transport of virions according to Labuda (Labuda et al., 1998) is performed by Langerhance cells and neutrophyles travel from one inflammatory focus in the place of attachment to another one.
VI - omovampiric way (Fig. 4, VI) of virus exchange between a starved female and infected male I. persulcatus in the period before or after copulation is quite probable, because we have proven that saliva of starved males contains a fairly large amount of virus, sufficient for infecting not only animals (Alekseev, 1991), but also man (Penjevskaja, 1989) and that feeding of males on females with which they later copulate can be observed in 2-10% cases. All the ways virus circulation constitute a powerful mechanism of homeostasis, stabilization of existence of triple parasitic system in the external environment. Changes of its conditions, e.g. temperature, strengthens the role of one way or another. Thus, development of eggs in infected female under temperate temperatures leads to the increase of the portion of individuals which obtained virus transovarially (Kondrashova, Filippovets, 1970); increase of summer temperature increases the frequency of occurrence of females with traces of male bites: occurrence of traces of bites is higher among southern populations of I. persulcatus (up to 10% in the region of Zhiguli) and is lower among northern ones (less than 2% in the Krasnoyarsk population) (Alekseev, 1991). Therefore under changing abiotic conditions different homeostasis mechanisms become engaged.
As was shown before (Alekseev, 1993), tick-pathogen represents a certain system possessing new properties, which we termed after Odum (1975) newly emergent properties. Thus, the presence of virus stimulates moving activity of females and their responses to attractants of animal origin, strengthens their negative geotaxis and weakens their positive humidity taxis (Alekseev et al., 1988a, 1991a), which leads to 7-10 fold increase of occurrence of infected females on man (Alekseev, Dubinina, 1994; Melnikova et al., 1996). The epidemiological importance of this phenomenon is obvious. Less obvious is the epizootological significance: infected females more actively attacking a large animal host increases thereby not only the probability of engorging and leaving of progeny, but also increases chances of retaining of micropopulation of virus in case of transovarial transmission. The increase of virus concentration in ticks increases their activity (Alekseev et al., 1996b). Positive correlation between factors appears: high intensity of infection - greater probability of obtaining of blood, higher level of infection - higher probability of laying a larger number of infected eggs (Kondrashova, Filippovets, 1970). It is highly probable, which can be tested, that more intensively infected females are more tolerant to temperate temperatures and the difference in activity from the uninfected ones will be even greater and this can increase even more the probability of virus transovarial transmission.
Our scheme and data accumulated up to now permit us to analyze the basis of stability also of other tick-borne infection: tick borrelioses (Lyme disease in the USA) vectors of which are ticks of the genus Ixodes and in Eurasia the same species as for tick-borne encephalitis - I. ricinus and I. persulcatus.
I - transovarial transmission of Borrelia was proven for the American species of the genus Ixodes I. scapularis. It has low efficiency in Ixodes pacificus Coley et Kohls and in European I. ricinus, but is rejected for I. persulcatus (Nakao, Miyamoto, 1992); transphasic transmission has been shown many times; also in I. persulcatus from nymphs to imago its was proven by E.I. Korenberg and co-authors (1988).
II - transmissive way has been proven many times and is undoubtedly the basic one, because many rodents are infected with Borellia for a lifetime.
III - we have recently proven sexual transmission and demonstrated the probability of omovampiric way of exchange of the pathogen (Alekseev, Dubinina, 1996a).
IV - transsalival and distant exchange of Borrelia between jointly feeding preimago of ticks I. ricinus has been also proven recently (Gern, Rass, 1996).
It appears therefore that there are no principled differences in the functioning of both ixodid tick infections. However they exist and can be of principle importance when the vector is subject to dual infections.
The system tickBorrelia functions in a different way than the system tick-virus: presence of Borrelia suppresses their moving activity (Alekseev, Dubinina, 1994), apparently to such extent that infected ticks occur on deer nearly 4 times less frequently, than on vegetation (Lacombe et al., 1992).
This difference made us think about a possible nature of interactions of two infections using for their support and dispersal the same vectors. In fact, if the rope is so strong, why only a part of the population of taiga ticks is infected with the TBE virus? If the schemes of circulation of two pathogens are so similar why borreliae do not occur in all ticks? And eventually, why mixtinfections are relatively rare?
American scientists have accumulated a large body of factual evidence on occurrence of borreliae and babesiae, borrelia and erlichiae and detected quite recently also virus of the tick-borne encephalitis group. However the question of interaction of these systems was not posed. Facts on the portion of mono- and mixtinfections were only stated. The question of why far not all mixtinfecitons are transmitted was not even posed. Meanwhile mixtinfections, seem to be a rule rather than exception.
In essence, the world of microorganisms in arthropods is a whole universe, living according to the intricate laws of interaction, a universe whose structure E.N. Pavlovsky has justly denoted parasitocosmos.
Specialists are interested in transmissive infections transmitted by blood-sucking arthropods are interested in those laws of functioning of this universe, which promote (or hamper) transmission of agents pathogenic for man and animals: viruses, microorganisms, protozoans.
Symbiosis, presence of commensals, mutualists, interaction of different pathogens in parasitocoenoses of intestine in human tissues is not surprising. However conceptions of parasitocoenoses of the intestine and organisms as a whole in invertebrates - hosts of pathogens about existence and functioning of their parasitocosmos, about its possible role in formation and maintaining of foci of transmissive infections have been elaborated insufficiently. Meanwhile in the organism of invertebrate host agents of transmissive diseases exist in an intricate complex; their successful development, infection of the vertebrate host and transmission in the course of metamorphosis to progeny to large extent depends on the presence of other members of the biocoenosis.
We have got accustomed to the notion of the pyramid of living things at the base of which the most numerous groups of procaryots and viruses are situated. Arthropods are situated at the highest level of the pyramid, their number is smaller and the number of vector species, bloodsucker species is very small as compared to other groups. Therefore the number of host species and their populations because of this definition cannot be lower than the number of species of microorganisms pathogens of diseases. We are no longer surprised by the fact that the same arthropods are vectors of pathogens of several infections. Following V.N. Beklemishevs (1970) definition of a transmissive disease focus as a population of pathogen together with populations and vectors maintaining their existence we inevitably come to the notion of polycausal foci where the same vectors participate in circulation of several pathogens. For instance, in Russia the same species of ixodid ticks transmit viruses (virus of tick-borne encephalitis) and bacteria Borrelia (Lyme disease pathogen) to man. This is not surprising: blood-sucking arthropods transmitting the pathogen for which according to the theory developed by us earlier (Alekseev, 1985) there is no prohibition can simultaneously transmit also any other permitted pathogen. It appears that ixodid ticks in question can transmit simultaneously rickettsiae, ehrlichiae, viruses, borreliae, pyroplasmids, babesiae and some filariae (Alekseev, 1993).
However this does not happen. Incidence of borrelioses and tick encephalitis is an exception rather than a rule. The relative rarity of mixtinfections occurring in humans is attributed by some authors simply to isolation of populations of taiga ticks infected by different pathogens (Korenberg et al., 1990). Comparison of literature data with the results of our field observations permits us not only to disagree with this opinion, but also to assume that functioning of the triple system with two pathogens in one vector is unstable, giving advantages to spirochetes as compared to virus.
The relationships of the number of ticks with mixtinfection and the number of ticks containing borreliae appeared to be 10-11 times lower than the relationship between tick number and dual infection and the number of ticks containing virus (Korenberg and others, 1990; our observations in Sayany). These data assuming that both pathogens occur in the same foci (all our own and literature data suggest this) can be explained only by the fact that for some cause borreliae have considerable advantage for penetrating ticks, for their preservation in populations and possible for transmission to vertebrate hosts and retaining them in the organisms as reservoir of infection.
Evidently, the nature of interaction of pathogen and vector determines both stability of parasitic system, and the very existence of the transmissive infection focus and its epidemical manifestations.
Our innovative approach to the elucidation of functioning of the focus of mixed infection is that the tick pathogen is considered as a new system, which is more complicated than uninfected tick, and characteristics of functioning of such system are studied (Alekseev, 1993).
We chose moving activity of ticks collected in nature as one of the main markers of functioning of the system and compared it in ticks, uninfected and infected by different pathogens and with different intensity. Used as a second criteria was susceptibility of ticks to one of the pathogens if the second is present and ability of reproduction of the 1st pathogen to different levels in specimens infected or uninfected by the 2-nd pathogen. Third criterion - influence of physiological age of ticks on the first two parameters.
All the three criteria are quantitative and are, therefore, subject to statistical processing.
We have already mentioned that unlike TBE virus, presence of Borrelia in the tick organism suppresses their moving activity, and made a supposition supported so far by the only observation on the ticks from Sayany (only one of several hundred individuals was infected at the same time by virus and borreliae) that in case of mixtinfection (virus+borreliae) moving ability of the vector can increase (Alekseev et al., 1996b). When ticks were kept in laboratory at a temperature of 20.0 - 1.5oC and with depletion of their food resources, indices of moving activity of vectors were gradually decreasing. Inhibition of moving activity of ticks infected with borreliae was increasing much faster than in case of normal physiological ageing (Alekseev et al., 1996b) of uninfected individuals. There is a reason to assume that this process is more intensive in ticks with dual borrelioses infection with carrying at the same time of two species of borreliae. Dual infection by borreliae of species Borrelia afzelii and Borrelia garinii is apparently typical of ticks I. persulcatus from the vicinity of St. Petersburg, where we have been conducting regular observations for the past 5 years (Alekseev et al., 1998). The sensitivity of ticks not infected by borreliae to TBE virus (Alekseev, 1998) repeats at a higher level the curve of borreliae prevalence in infected ticks during the season (Fig. 5, 1) and declines with the increase of the portion of physiologically older ticks in a population (data on physiological age are used after Balashov, 1962). Ticks infected with borreliae appeared to be 2.5 times less sensitive to intracoelomal injection of virus, and the virus reproduced up to high titres (higher than 1.5 lg LD50 in 0.03 ml in case of intracerebral infection of the new-borne suckling mice); virus was reproduced 1.5 times less frequently than in ticks free from borreliae. Sensitivity of ticks infected with borreliae to TBE virus (Fig. 5, 2) falls parallel to the abundance of individuals with monoinfection (Fig. 5, 3) and to the decrease of the number of individuals of the 2nd physiological age (Fig. 5, 4). A positive correlation, according to Pearson, between these parameters is equal to +0.922. The maximal level of correlation of sensitivity of ticks with borreliae to TBE virus is observed when ticks of the second physiological age are predominant in the season, whereas predominance of ticks infected with borreliae and sensitivity of uninfected individuals to virus is correlated in the best way with maximal abundance in the season of ticks of the third physiological age (according to gradation introduced by Yu.S. Balashov, 1962).
Fig. 5. Prevalence of Borrelia-infection in Ixodes persulcatus ticks within a season and tick susceptibility of TBE virus infection.
1 – ratio of ticks of the II-nd physiological age in Leningrad region; 2 – ratio of ticks infected only by one Borrelia species (1995); 3 – ratio of Borrelia infected ticks susceptible to virus infection (1994); 4 – extensity of Borrelia-infection according dark-field microscopy data, St. Petersburg mean data of 1993-1996.
During the season not only sensitivity of borreliae infected ticks to virus, but also levels of TBE virus reproduction in individuals that received it were decreasing. One has to elucidate whether borreliae cause premature ageing of ticks depleting their food supply, or they change structures of receptor fields of membranes of tick cells and their ability to virus reproduction. However it is clear already now that the environment tickborreliae, particularly in case of dual infection (according to preliminary data received at 1995 more than a half of I. persulcatus of St. Petersburg population were infected with B. afzelii and B. garinii) is unfavourable for TBE virus reproduction. It is curious that correlations between number of borreliae in ticks mid-gut (250 fields of vision in the dark field - that is the way intensity of their infection is determined) and their sensitivity to TBE virus are absent. The lack of relationship between sensitivity of ticks to virus and intensity of infection of imago by borreliae suggest that changes in metabolism and physiological status of ticks providing for peculiar features of their response to virus occur at preimaginal phases of development of these bloodsucking arthropods, i.e. on the phase of larva or nymph when infection of I. persulcatus with borreliae takes place, because in all probability there is no transovarial transmission in this tick species (Nakao, Miyamoto, 1992).
In the case analysed by us we definitely come across antagonism of two pathogens using as a vector the same species of ticks. Specimens infected by borreliae as we mentioned before are 2.5 times less perceptible to parenteral infection by highly virulent strain of TBE than specimens in which borreliae were not detected by methods used. However all processes of antagonistic interaction borreliaevirus take place at preimago stages and go much faster because specimens infected by borreliae as a rule predominate in the vector population. Mixts among ticks with borreliae occur 10 times less frequently, than mixts among ticks with virus. It follows that occurrence of virus in specimens with borreliae is hampered by not 2.5 fold as it is observed in laboratory, but 10-11 fold, as it was discovered in nature. Reproduction of virus in ticks with borreliae is also hampered. Possibly this particular factor is responsible for a milder virus infection with a simultaneous infection by both pathogens or lack of symptoms of TBE in patients sick with borreliosis or in case of presence of virus in ticks, the bite of which caused disease. Thus only in 4.8% of 249 cases in Sverdlovsk Region encephalitis was serologically identified as TBE (Lykovskaya et al., 1993). Based on our experimental data and on results of survey of epidemiologists one can say with a fair degree of assurance that successful and unrestricted circulation of TBE virus in natural foci is hampered by competition of borreliae. The external manifestation of this phenomenon is the change of perceptibility to virus and moving activity of ticks, similar to one observed in individuals not infected by borreliae during normal physiological ageing.
Does the presence of virus in the focus of borreliae circulation pose an obstacle? We have no straightforward experimental data, however analysis of some indirect indices permits us to give a negative answer. Firstly, among ticksvirus carriers, mixts occur more frequently, than among ticks, infected with borreliae. Secondly, among patients with tick-borne encephalitis borreliosis (or antibodies to borreliae) are discovered at least 3.5 times more frequently than TBE symptoms or antibodies to TBE virus among patients with borreliosis (16.7 as compared to 4.8. in the same Sverdlovsk Region (Lykovskaya et al., 1993). Thirdly, probably because penetration of borreliae in the virus infected tick eases the impact of borreliae suppressing its moving activity.
Functioning of the focus of mixt tick infections represents, in our opinion, a peculiar form of one-sided antagonism when circulation of borreliae restricts circulation of TBE virus, whereas presence of virus in ticks not only does not hamper but probably even promotes transition of Borrelia. Possible explanation of these differences is that considered tick pathogens use different phases of vectors cycle for circulation in the system tick - warm blooded host. Whereas TBE virus uses all phases of the cycle from egg to imago and transovarial transmission is needed for its preservation, it is sufficient for the circulation of borreliae to have exchange of pathogen between nymphs and larvae of ticks feeding on the same small mammals, that retain borreliae in their organism nearly for lifetime (Lane et al., 1991). The indirect evidence is occurrence frequency of two species of Borrelia in adult ticks of I. persulcatus.
For a successful transovarial transmission successful and complete engorgement of imago and their fertilisation is necessary. Stimulation of vectors moving and searching activity increases the probability of preservation of virus populations and their genetic constancy. Large concentration of virus stimulates activity of females and males.
One can assume that the absence of correlation between moving activity (and sensitivity to virus) and intensity of imago infection, as well as the low probability or total absence of transovarial transmission of borreliae is the manifestation of blind alley of the circulation of borreliae during their transition into imago.
Feeding of Ixodes females on large animals does not provide for transition of this pathogen (Gill et al., 1993; Gray, 1991). Most probably competitive interactions are revealed at the preimago phase and depend on the sequence of penetration of one or other pathogen in the vector; larvae that have been infected with virus transovarially are easily infected by borreliae during feeding on rodents - reservoirs of this pathogen, whereas infection with virus of nymphs already infected by borreliae is hampered considerably.
It is extremely curious, that at least in some very rare cases (Chernogor et al., 1996) presence of TBE virus in tick during mixtinfection of man can block the action of antibiotics on spirochetes, usually highly sensitive to doxicycline.
It appears to us that the ecological approaches to the consideration of tick vectors as to habitat changed by one pathogen for another is highly promising and contain innovative features typical of Russian science. The analysed interaction of two systems tickBorrelia, tickBorreliavirus represents the first in the worlds science evidence of antagonism of viral and bacterial agents. Elucidation of these mechanisms may become an impetus to application of totally new membrane- cell- defence- antiviral- mechanisms, a topical phenomenon in the era of AIDS.
Moreover, the consideration of the functioning of the system tickBorrelia under conditions of intensive anthropogenic pressure can serve as an example of using epidemiological significant acarological markers to predict trends of development of harmful consequences of mans technogenic activities.
For instance, accumulation of ions of heavy metals in the vicinity of large cities and changes of chitinous structures of arthropods related to them occurring, as we have shown in the experiment, after the increase of ion concentration (Dubinina, Alekseev, 1994) involves not only soil, oribatid mites, but also blood-sucking ticks ixodids - vectors of diseases (Alekseev, Dubinina, 1993). The number of comparatively slight changes (that cannot be regarded as abnormalities proper) in the exoskeleton of the main vector of tick infections in Russia - tick I. persulcatus was the greater, the higher anthropogenic pressure could be assumed for the studied territory. The largest number of such changes was discovered in the vicinity of Vologda affected by the Cherepovetsky Metallurgic Complex. Ranking next to this area in the number of changes was the vicinity of St. Petersburg (Alekseev, 1995; Alekseev, Dubinina, 1996d, 1997).
We have established that pathological changes in the integument of ticks are accompanied by the changes of behavioural responses. At the same time we have noted similar changes of behaviour also under the influence of presence of pathogens of tick borrelioses (Alekseev, 1996b), in particular B. afzelii and B. garinii circulating in the foci of these diseases in the vicinity of St. Petersburg. There is reason to assume that in case of infection by two borreliosis agents simultaneously suppression of moving activity of ticks is essentially higher. The similarity of phenomena permitted us to assume that the anomalies of integuments being the reflection of biochemical and physiological rearrangements in ticks organism as a result of possible substitutions of calcium salts (Norton, Behan-Pelletier, 1991), participating in the formation of exoskeleton of ticks, by salts of other metals can lead to changes in interaction in the system tick pathogen - tick (Alekseev, 1993).
Average abundance of ticks with borreliae fluctuates during the season from 11% to 30%, average number of specimens with anomalies of chitinous covers also varies during the season: from 15% to 63% in some 10 day periods (observations of 1993-1994, Alekseev, 1995) (Fig. 5), but it is considerably higher than in normal ticks in the middle of the season of tick activity (Fig. 6).
Fig. 6. Prevalence of ticks with exoskeleton anomalies in the St. Petersburg vicinity.
Comparison of curve A in Fig. 6 and curve 3 in Fig. 5 also permits us to suppose that in ticks with pathology the maximum of borreliae occurrence i.e., end of May - beginning of June, coincides with a sharp increase (by more than 50%) of occurrence of ticks infected simultaneously by two species of Borrelia. Over the past 3 years number of ticks with pathologies of chitin decreased in the vicinity of St. Petersburg 1.5 fold as compared to 1992-1993 (Fig. 7), which probably was related to the decrease of technogenic pressure (e.g. stagnation of military industrial complex). In the beginning of observations the number of Pb and Ni ions in the vicinity of the zone where ticks were collected exceeded the sanitary standards by two orders of magnitude. At present, the situation has probably slightly improved. However from 1993 through 1995 the tendency of increase of borreliae infection of ticks with chitin pathology as compared to the infection of normal ticks remained (Fig. 8).
Fig. 7. Prevalence of Borrelia infected Ixodes persulcatus within a season.
A – anomalous ticks; N – normal ticks.
Fig. 8. Correlation of prevalence of Borrelia infected ticks with (A) and without (N) anomalies.
The impact of environment anthropogenic transformation on the growth of Lyme disease incidence in man has already attracted attention of researchers. This phenomenon was attributed in America to restoring of forests and increase of deer livestock (Matuschka, Spielman, 1986). This interpretation is in no way adequate for the conditions of Eastern Europe and Russia. Forest areas and abundance of large ungulates are decreasing, and the growth of environmental pollution, primarily by ions of heavy metals causes, justified alarm of all ecologists (Feshbach, Frendly-Jr., 1992).
One of the many consequences of environmental pollution and transformation as a result of mans activity is the circulus vitiosus: growing anthropogenic pressure - appearance of anomalies in vectors - related to these increase of the level of infection of the main vector by borreliae - the increasing incidence with tick borrelioses noted by epidemiologists and practitioners.
The case considered is one of the many examples of the systematic approach to numerous and grave ecological problems faced by our country in this century. It seems to us that the consideration of functioning of parasitic biological systems as a totality of subsystems with new properties arising with their complication and with refining of our understanding of their structure and interaction will permit us not only to refine our knowledge on the vicious circles arising as a result of mans activity, but to corroborate new concepts in the field of ecological parasitology and parasitocoenology.
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