Abstract: This book presents the state of art in the field of microbial zoonoses and sapronoses. It could be used as a textbook or manual in microbiology and medical zoology for students of human and veterinary medicine, including Ph.D. students, and for biomedicine scientists and medical practitioners and specialists as well. Surprisingly, severe zoonoses and sapronoses still appear that are either entirely new (e.g., SARS, Hendra and Nipah viroses, hantavirus pulmonary syndrome), newly recognized (Lyme borreliosis, ehrlichiosis, anaplasmosis), resurging (West Nile fever in Europe), increasing in incidence (salmonellosis after 1988, campylobacterosis), spatially expanding (West Nile fever in the Americas), with a changing range of hosts and/or vectors, with changing clinical manifestations or acquiring antibiotic resistance. The collective term for those diseases is (re)emerging infections, and as much as 75% of them represent zoonoses and sapronoses (the rest are anthroponoses). The number of known zoonotic and sapronotic pathogens of humans is continually growing â over 800 today.
In the introductory part, short characteristics are given of infectious and epidemic process, and the role of environmental factors in the spread of zoonoses and sapronoses, possibilities of their epidemiological surveillance, and control. Much emphasis is laid on ecological aspects of zoonoses and sapronoses (haematophagous vectors of diseases and their life history; vertebrate hosts of zoonosesï» habitats of the agents and their geographic distributionï» natural focality of diseases). Individual zoonoses and sapronoses are then characterized in the following brief paragraphs: taxonomy and nomenclature; source of human infection; animal disease; transmission mode; human disease; epidemiology; diagnostics; therapy; geographic distribution (accompanied with maps).
Abstract: Cryopreservation of microorganisms below -140 C (ensuring that any biochemical and cryogenic processes are stopped) is the best method for a safe, long-term maintenance in microbial technology (production strains) and culture collections (reference or type strains). It usually retains high viability as well as phenotypic and genomic stability of the preserved cultures. All sorts of microorganisms (viruses, bacteria, fungi, microalgae and protozoa) have been treated comprehensively. The bibliography includes more than 1,000 references on the topic. The book might be a relevant reference guide to cryopreservation for microbiologists in culture collection, industry, biotechnological laboratories, genetic engineering, preventive medicine and additional biological disciplines.
Abstract: Abstract To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.
Abstract: Host-seeking Dermacentor reticulatus ticks were detected by flagging method at 46 localities at south-east part of the Czech Republic, in the basins of rivers Morava and Dyje. Exact north-west distribution limits of D. reticulatus were defined in this area for the first time. Detailed prediction map of probabilities of D. reticulatus occurrence was obtained using GIS analysis. Spatial model delimited a south-north gradient in probability across the studied area, with highest probabilities above 0.8 in its southernmost part. Abundance of D. reticulatus varied markedly between localities in interval 0.33-222 of ticks per flag per hour. The highest abundances were in flooded areas at lower streams, towards upper streams abundance and density of these ticks decreased. Females prevailed in samples with population sex ratio of 0.413, significantly deviating from parity. Larvae and nymphs of this species were not detected by flagging. Although D. reticulatus range expansion probably did not reach such a degree as reported in other countries, these ticks became very abundant in some parts of studied area. Since spreading of vector-borne diseases became a problem in Europe, the knowledge of their exact recent geographic ranges is important for future modelling of their shift predictability.
Abstract: A total of 1935 migratory birds from 104 different species were captured in southeastern Sweden in 2005-2006 and tested for antibodies against West Nile virus (WNV). Overall, 46 birds (2.4%; binomial confidence limits, 1.8-3.2) were positive by blocking-ELISA, but only 2 (0.10%; binomial confidence limits, 0.0-0.4) had antibodies detectable by both blocking-ELISA and WNV neutralization test. ELISA-positive birds included long- and short-distance migrants likely exposed to WNV while wintering in or migrating through areas enzootic for WNV. Exposure to a cross-reactive Flavivirus was suspected for short-distance migrants of the Turdidae family, but no cross-neutralization with tick-borne encephalitis and Usutu viruses was observed.
Abstract: A serosurvey for West Nile virus (WNV) was carried out in 525 persons, using a plaque-reduction neutralization microtest with Vero cells and Egyptian topotype Eg-101 strain as test virus. The blood sera were sampled in four South-Moravian districts (HodonÃn 44 persons, BÅeclav 102 persons, Znojmo 170 persons, Jihlava 209 persons) of the Czech Republic in the years 1988 and 1989, and stored at -20 ºC since the collection. Antibodies to WNV were detected in only three humans (0.6% seropositivity): one person each in the districts of HodonÃn (2.3% persons positive), BÅeclav (1.0% positive) and Jihlava (0.5% positive), with the titres of 1:64, 1:32, and 1:32, respectively. All the three sera were negative for antibody to tick-borne encephalitis virus. The results indicate that activity of WNV in southern Moravia was very low before 1990.
Abstract: The main goals of the study were to carry out virus isolation attempts on Vero cell cultures from mosquitoes collected in southern Moravia (Breclav district, Czech Republic) and to identify the isolates using a microtiter virus neutralization test. A total of 9.742 female mosquitoes belonging to 13 species were examined, and three viral strains were isolated, all from the mosquitoes collected in 2006: two of these isolates were identified as Tahyna Orthobunyavirus (both obtained from Aedes vexans mosquitoes) and one was West Nile flavivirus (obtained from Aedes rossicus mosquitoes). Ae. rossicus might be a new vector for West Nile virus.
Abstract: In the last 30 years several cases of West Nile (WN) virus infection were reported in horses and humans in Europe and in the Mediterranean Basin. Most of them were determined by strains of the Lineage 1 included in the European Mediterranean/Kenyan cluster. Strains of this cluster are characterised by a moderate pathogenicity for horses and humans and limited or no pathogenicity for birds. In recent years, however, WN cases determined by strains grouped in the Israeli/American cluster of Lineage 1 or in the lineage 2 have been reported in Hungary and Austria. The role of migrating birds in introducing new viruses to Europe has been often demonstrated. The migratory birds, which may be infected in their African wintering places, carry the virus northward to European sites during spring migrations. In the past, the virus introduction determined occasional cases of WN. In the recent years, new epidemiological scenarios are developing. In few occasions it has been evidenced the capability of WNV strains of overwintering by using local birds and mosquitoes. Species of Culex amongst mosquitoes and magpies (Pica pica), carrion crows (Corvus corone) and rock pigeons (Columba livia) amongst resident birds are the most probable species involved in this hypothetical WND endemic cycle.
Abstract: An update on the mosquito-borne flavivirus species including certain subtypes, as listed in the Eighth Report of the International Committee on Taxonomy of Viruses, is given. Special emphasis is placed on viruses which have been shown to cause diseases in animals, and viruses for which no pathogenicity has been proven yet. Several recent examples (Usutu virus and lineage-2 West Nile virus in central Europe, Zika virus in Micronesia) have shown that sources providing information on such scientifically largely neglected viruses are valuable tools for scientists and public health officials having to deal with such disease emergences. Furthermore the effects of global warming will lead to introduction of competent mosquito vectors into temperate climate zones and will increase efficiency of viral replication in less competent vector species. This, facilitated by rising global travel and trade activities, will facilitate introduction and permanent establishment of mosquito-borne viruses, some of which may become of public health or veterinary concern, into novel environments, e.g. industrialized countries worldwide.
Abstract: Six viral isolates were obtained from 23,243 female mosquitoes (examined in 513 pools) belonging to 16 species and collected along the lower reaches of the Dyje River in South Moravia (Czech Republic, central Europe) during 2006-2008: five isolates of Orthobunyavirus ŤahyÅa (TAHV, California group, family Bunyaviridae): 3 isolations from Aedes vexans Meigen, 1 from Ae. sticticus (Meigen), 1 from Culex modestus Ficalbi), and one isolation of Flavivirus West Nile (WNV, Japanese encephalitis group, family Flaviviridae) â strain Rabensburg (proposed lineage 3 of WNV) from Aedes rossicus Dolbeshkin et al. All viral isolates were recovered from mosquitoes collected in 2006 (15,882 mosquitoes examined), while no virus was isolated from mosquitoes trapped in 2007 and 2008, when 1,555 and 5,806 mosquitoes were examined, respectively. The population density of local mosquitoes was very low in 2007 and 2008 due to warm and dry summer including a considerably low water table, compared to environmental conditions favorable for mosquito development in 2006. The virus isolation procedure was based on intracerebral inoculation of newborn mice. In parallel, more than one-third of the samples (183 pools consisting of 8470 individual mosquitoes) were also examined by inoculating Vero cell cultures in Leighton tubes. However, the latter method detected only 3 of the 6 virus isolates (including WNVâRabensburg). Aedes rossicus is a new potential vector for WNVâRabensburg. This species feeds mostly on mammals including man; this raises the question whether this virus lineage is adapted to an alternative mosquitoâmammal cycle in the South-Moravian natural focus.
Abstract: Mosquito collections with CDC light traps using dry ice and pigeon-baited traps were carried out in south Moravia (Czech Republic) from April to October in 2007 and 2008 at two study sites. In 2007, 11 two-day captures were carried out in two-week intervals, and 1,490 female mosquitoes of nine species were caught. In 2008, 15 two-day trappings of mosquitoes were carried out: 6,778 females of 22 species of mosquitoes were trapped. The results showed marked differences in abundance and species composition of mosquitoes between both study sites and between the trapping methods. In the floodplain forest ecosystem of the Soutok study area, Aedes vexans predominated. The species composition in the Nesyt study site was more varied and the most common species was Culex pipiens. At the latter study site, Anopheles hyrcanus (var. pseudopictus) and Uranotaenia unguiculata, mosquito species with largely southern Eurasian distribution, were repeatedly demonstrated. The largest capture of mosquitoes was in traps with CO(2) placed at a height 1 m above the ground. The capture of mosquitoes in the pigeon-baited traps as well as in the traps with CO(2) placed in the canopy of trees was markedly lower in both study sites, with the predominant species being Culex pipiens.
Abstract: Two strains of Gram-positive cocci were isolated from viscera of common voles, Microtus arvalis (Pallas), with generalized Brucella microti infection in the Czech Republic. Biochemical features and phylogenetic analysis based on 16S rRNA gene sequences proved that the strains are representatives of the genus Staphylococcus and assigned Staphylococcus muscae as next relative. A detailed characterization done by ribotyping, rpoB and hsp60 gene sequencing, whole-cell protein analysis and rep-PCR using the (GTG)(5) primer differentiated the two strains from all validly described staphylococci. DNA-DNA hybridization with the type strain of S. muscae demonstrated that the two strains should be considered as a novel species (26.8% reassociation). Two analysed strains were found to be coagulase-negative, novobiocin-susceptible, oxidase negative cultures phenotypically close to one another, but showing differences in ribotype profiles. The major fatty acids were iso-C(15:0), iso-C(17:0) and anteiso-C(15:0), unsaturated C(18:2)omega6,9c/ante-C(18:0) and C(18:1)omega9c. MK-7 and minor amounts of MK-6 and MK-8 were found as the predominant isoprenoid quinones. The polar lipid profile is composed of the major lipids diphosphatidylglycerol and phosphatidylglycerol and several unknown lipids. The obtained results proved that the two isolates are representing a novel staphylococcal species. The name proposed for this novel taxon is Staphylococcus microti; the type strain is CCM 4903(T) (=CCUG 55861(T)).
Abstract: A total of 178 free-living birds of 14 species of 7 families of Passeriformes sampled in a freshwater reedswamp habitat in southern Moravia in July 2006 were examined for hemagglutination-inhibiting (HI) antibodies to Alphavirus Sindbis (SINV), and bunyaviruses Tahyna (TAHV) and Batai (BATV). Hemagglutination-inhibiting antibody was detected against all three viruses, but at different frequencies: SINV 0.7%, TAHV 14.0%, and BATV 6.8%. The survey indicates circulation of mosquito-borne viruses TAHV and BATV and very low, if any, SINV activity in the area.
Abstract: Sera of 642 wild boars (Sus scrofa) shot by hunters in ten administrative regions of the Czech Republic during 1995â2000 were tested by indirect haemagglutination assay (IHA) for the presence of anti-Borrelia IgG. Antibodies to Borrelia burgdorferi sensu lato (Bb) were detected in sera from all 10 regions, and overall seroprevalence was 12.8%. Titres of antibodies ranged from 1:80 to 1:640. Anti-Borrelia antibodies were most frequent in the wild boars from three administrative regions of the Czech Republic: Moravskoslezský (25.0%), Pardubický (25.0%) and Královehradecký (24.1%), followed by the regions PlzeÅský (16.7%), Olomoucký (13.3%), Jihomoravský (12.8%), VyÅ¡kovský (11.1%), JihoÄeský (11.1%), ZlÃnský (10.3%) and Liberecký (8.9%). Seasonal seroprevalence increased in March and April, the peak was in May. The results suggest an increased extent of wild boars' exposure to Bb in predominantly rural and forested regions. The study also reviews the importance of wild boar in Lyme borreliosis (LB) ecology. Wild boars serology may provide another means of surveillance of areas with human risk for LB.
Abstract: In 2005 and 2006, Ixodes ricinus ticks were collected on two slag (waste rock) heaps from coal mines in the Ostrava area (North Moravia/Silesia, Czech Republic), Oskar (site A) and Emma (site B), partially covered by vegetation including trees, and at a control forest site near HlucÃn (site C). The mean numbers of L. ricinus nymphs and imagoes flagged per person-hour were high: 35.3 nymphs and 12.7 imagoes, at site A, 23.3 and 26.0, respectively, at site B, and 25.4 and 16.8, respectively, at control site C. Using dark-field microscopy, 100 nymphs and 100 imagoes (50 females and 50 males) from each site were examined for borreliae. The mean prevalence rates of Borrelia burgdorferi sensu lato in nymphs and imagoes were 10.0% and 12.0%, respectively, at site A, 10.0% and 24.0%, respectively, at site B, and 13.0% and 17.0%, respectively, at site C. Differences in the prevalence of borreliae in nymphal and adult ticks from the slag heaps and control site were insignificant, but adult ticks from site B compared to site A contained borreliae significantly more frequently. The mean numbers of nymphs and imagoes infected with borreliae flagged per person-hour were 3.3 and 1.2, respectively at site A, 1.5 and 2.9, respectively, at site B, and 3.1 and 2.6, respectively, at site C. Isolation experiments for borreliae were carried out only in 16 ticks containing higher numbers of borreliae, with eight of these being culture-positive. The cultured borreliae were identified by PCR-RFLP as B. garinii (3 isolates: two from site B, one from site C), B. afzelii (4 isolates: one from site A, three from site B) and B. burgdorferi s.s. (one isolate from site A). Surprisingly, the results suggest that slag heaps, when covered by woody vegetation and frequented by humans, could theoretically pose roughly the same LB transmission risk to humans as common forest biotopes.
Abstract: Lung tissue of 156 rodents from the Czech Republic (genera Apodemus, Myodes, Microtus, Muscardinus), 29 rodents Spalax ehrenbergi from Israel and 106 from Africa (genera Heliophobius, Mastomys, Acomys, Aethomys, Saccostomus, Tatera , Mus, Fukomys , Dasymys, Dendromus, Grammomys, Steatomys) was examined for presence of adiaspores of the fungal genus Emmonsia. In Czechland, nine (5.8 %) animals were positive. The positive samples were found in rodents sampled in three of the five areas studied. Out of six species of Czech rodents, three spp. were positive: Apodemus flavicollis was the most frequently infected species, whereas A. sylvaticus, Microtus subterraneus and Muscardinus avellanarius were negative. Adiaspores were recorded in five females and four males. Tissue samples of rodents from Africa and Israel were all negative.
Notes: A related paper: Hubálek Z., Burda H., Scharff A., Heth G., Nevo E., Šumbera R., Peško J., Zima J. (2005): Emmonsiosis of subterranean rodents (Bathyergidae, Spalacidae) in Africa and Israel. Med. Mycol. 43: 691-697.
Abstract: Bhanja virus (BHAV) is pathogenic for young domestic ruminants and also for humans, causing fever and affections of the central nervous system. This generally neglected but veterinary important arbovirus of the family Bunyaviridae is transmitted by metastriate ticks of the genera Haemaphysalis, Dermacentor, Hyalomma, Rhipicephalus, Boophilus, and Amblyomma. Geographic distribution of BHAV covers southern and Central Asia, Africa, and southern (partially also central) Europe. Comparative biogeographic study of eight known natural foci of BHAV infections in Europe (in Italy, Croatia, Bulgaria, Slovakia) has revealed their common features. Based on the analysis, as 'predictors' of potential BHAV areas in Europe have been established following groups of factors: (1) submediterranean climatic pattern with dry growing season and wet mild winter (or microlimatically similar conditions, e.g. limestone karst areas in central Europe); (2) xerothermic woodlandâgrassland ecosystem, with plant alliances Quercetalia pubescentis, Festucetalia valesiacae, Brometalia erecti, involving pastoral areas on undulating to hilly relief with regular grazing of ruminants; (3) presence of at least one of the metastriate tick species Haemaphysalis punctata, Dermacentor marginatus, Rhipicephalus bursa and/or Hyalomma marginatum; (4) presence of â¥60% of the 180 species suggested as BHAV bioindicators (157 plant, 4 ixodid tick, and 19 vertebrate spp.). On that basis, Greece, France (southern, including Corsica), Albania, Spain, Hungary, European Turkey, Ukraine (southern), Switzerland (southern), Austria (southeastern), Germany (southern), Moldova, and European Russia (southern) have been estimated as additional European regions where BHAV might occur.
Abstract: During a survey of mosquitoes in the South Moravian lowland area, the mosquito Anopheles hyrcanus (Pallas) (Diptera: Culicidae) was found breeding in an ancient fishpond (Nesyt). It is not clear whether this southern Palaearctic species, a known vector of malaria in Asia which has not been recorded in the Czech Republic until this year, has gone undetected in the past or whether it has recently moved into the region as a result of climate change.
Abstract: Blood sera collected from 400 domestic animals (260 cattle, 100 Merino sheep, and 40 Hutzul horses) in northeastern Hungary in 2005 were examined for antibodies against two tick-borne viruses, tick-borne encephalitis flavivirus (TBEV) and Bhanja bunyavirus (BHAV). Using ELISA as screening test and plaque-reduction neutralization as confirmatory test, seropositivity to TBEV was found to be 26.5% in cattle, 7.0% in sheep, and 0.0% in horses. Among cattle, the animals up to 3 years old had significantly lower seroprevalence rate than those in older age groups. Natural foci of tick-borne encephalitis are obviously present in northeastern Hungary. On the other hand, no antibodies neutralizing BHAV were detected in the domestic animals.
Abstract: Lyme borreliosis (LB) is the most frequent ixodid tick-borne human disease in the world, with an estimate of 85,000 patients annually (Europe 65,000; North America 16,500; Asia 3,500; North Africa 10; approximate figures). This chapter summarizes the up-to-date knowledge on all facts and factors important in the epidemiology of LB over the world. Individual paragraphs describe briefly the causative agent(s) and clinical manifestations of LB; biological vectors and hosts; ecosystems and habitats of LB spirochetes; geographic (latitudinal and altitudinal) distribution and incidence rates of LB in individual countries; seasonal distribution; patient's age, sex, and profession; comparison of urban vs. rural settings; modes of LB transmission (ixodid ticks as biological vectors; haematophagous insects as possible occasional mechanical vectors; other potential modes); weather effects on LB incidence; risk factors for LB acquisition by humans, and risk assessment. The chapter finishes with the possibilities of prevention and recommends a better epidemiological surveillance of LB, including the morbidity notification in additional countries where it has not yet been implemented.
Abstract: Usutu virus (USUV), family Flaviviridae, has been responsible for avian mortality in Austria from 2001 to 2006. The proportion of USUV-positive individuals among the investigated dead birds decreased dramatically after 2004. To test the hypothesis that establishment of herd immunity might be responsible, serological examinations of susceptible wild birds were performed. Blood samples of 442 wild birds of 55 species were collected in 4 consecutive years (2003--2006). In addition, 86 individuals from a birds of prey rehabilitation centre were bled before, at the peak, and after the 2005 USUV transmission season in order to identify titre dynamics and seroconversions. The haemagglutination inhibition test was used for screening and the plaque reduction neutralization test for confirmation. While in the years 2003 and 2004 the proportion of seropositive wild birds was <10%, the percentage of seroreactors raised to >50% in 2005 and 2006. At the birds of prey centre, almost three quarters of the owls and raptors exhibited antibodies before the 2005 transmission season; this percentage dropped to less than half at the peak of USUV transmission and raised again to almost two thirds after the transmission season. These data show a from year to year continuously increasing proportion of seropositive wild birds. The owl and raptor data indicate significant viral exposure in the previous season(s), but also a number of new infections during the current season, despite the presence of antibodies in some of these birds. Herd immunity is a possible explanation for the significant decrease in USUV-associated bird mortalities in Austria during the recent years.
Abstract: A new species Brucella microti, isolated from ill common voles (Microtus arvalis) in southern Moravia in 2000, was described lately. Here we report the successful cultivation of B. microti directly from soil samples that have been collected on the same locality seven years after primary isolation. The particular metabolic capabilities of B. microti and its persistence in the soil over a long time-period indicate that soil might be the natural habitat of this pathogen of rodents.
Abstract: Tick-borne encephalitis (TBE), the most serious widespread vector-borne disease of humans in Europe, increased from 2- to 30-fold in many Central and Eastern European countries from 1992 to 1993, coinciding with independence from Soviet rule. Unemployment and low income have been shown in Latvia to be statistically associated with high-risk behaviour involving harvest of wild foods from tick-infested forests, and also with not being vaccinated against TBE. Archival data for 1970--2005 record major changes in the agricultural and industrial sectors, and consequent changes in the abiotic and biotic environment and socio-economic conditions, which could have increased the abundance of infected ticks and the contact of humans with those ticks. For example, abandoned agricultural fields became suitable for rodent transmission hosts; use of pesticides and emissions of atmospheric industrial pollutants plummeted; wildlife hosts for ticks increased; tick populations appear to have responded; unemployment and inequality increased in all countries. These factors, by acting synergistically but differentially between and within each country, can explain the marked spatio-temporal heterogeneities in TBE epidemiology better than can climate change alone, which is too uniform across wide areas. Different degrees of socio-economic upheaval caused by political transition in Estonia, Latvia, Lithuania, Slovenia and the Czech Republic can apparently explain the marked variation in TBE upsurge. Causal linkage between national socio-economic conditions and epidemiology is strongly indicated by striking correlations across eight countries between the degree of upsurge of TBE and both poverty and household expenditure on food (R2 = 0.533 and 0.716, respectively).
Abstract: Two Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains CCM 4915(T) and CCM 4916), isolated from clinical specimens of the common vole Microtus arvalis during an epizootic in the Czech Republic in 2001, were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA (rrs) and recA gene sequence similarities, both isolates were allocated to the genus Brucella. Affiliation to Brucella was confirmed by DNA-DNA hybridization studies. Both strains reacted equally with Brucella M-monospecific antiserum and were lysed by the bacteriophages Tb, Wb, F1 and F25. Biochemical profiling revealed a high degree of enzyme activity and metabolic capabilities not observed in other Brucella species. The omp2a and omp2b genes of isolates CCM 4915(T) and CCM 4916 were indistinguishable. Whereas omp2a was identical to omp2a of brucellae from certain pinniped marine mammals, omp2b clustered with omp2b of terrestrial brucellae. Analysis of the bp26 gene downstream region identified strains CCM 4915(T) and CCM 4916 as Brucella of terrestrial origin. Both strains harboured five to six copies of the insertion element IS711, displaying a unique banding pattern as determined by Southern blotting. In comparative multilocus VNTR (variable-number tandem-repeat) analysis (MLVA) with 296 different genotypes, the two isolates grouped together, but formed a separate cluster within the genus Brucella. Multilocus sequence typing (MLST) analysis using nine different loci also placed the two isolates separately from other brucellae. In the IS711-based AMOS PCR, a 1900 bp fragment was generated with the Brucella ovis-specific primers, revealing that the insertion element had integrated between a putative membrane protein and cboL, encoding a methyltransferase, an integration site not observed in other brucellae. Isolates CCM 4915(T) and CCM 4916 could be clearly distinguished from all known Brucella species and their biovars by means of both their phenotypic and molecular properties, and therefore represent a novel species within the genus Brucella, for which the name Brucella microti sp. nov. with the type strain CCM 4915(T) (=BCCN 07-01(T)=CAPM 6434(T)) is proposed.
Abstract: A serosurvey for West Nile virus (WNV) was carried out in 54 domestic birds (geese and ducks bred on fishponds) and 391 wild birds representing 28 migratory and resident species, using a plaque-reduction neutralization microtest with Vero cells and Egyptian topotype Eg-101 strain as test virus. The birds were sampled in the South-Moravian fishpond ecosystem between 2004 and 2006. Antibodies to WNV were not detected in domestic waterfowl, but 23 (5.9%) free-living birds of 10 species showed a positive response. These were the common coot (Fulica atra, 5 positive/18 examined), common kingfisher (Alcedo atthis, 1/1), reed warbler (Acrocephalus scirpaceus, 2/80), sedge warbler (A. schoenobaenus, 3/80), marsh warbler (A. palustris, 2/28), Savi's warbler (Locustella luscinioides, 3/12), reed bunting (Emberiza schoeniclus, 1/28), blackcap (Sylvia atricapilla, 2/11), penduline tit (Remiz pendulinus, 1/14), blue tit (Parus caeruleus, 1/1), and starling (Sturnus vulgaris, 2/4). The antibody titers were comparatively low (1:20-1:40), and the only high titer (1:160) was found in an adult marsh warbler. When 14 of the sera reacting with WNV were titrated in parallel with Usutu Flavivirus, 12 were interpreted as having specific antibodies to WNV, one coot had a higher titer against Usutu virus, and another one could not be attributed to either of the two viruses. In conclusion, 13 (3.3%) of 391 wild birds had specific antibodies to WNV. The results indicate that WNV activity in southern Moravia was limited during 2004-2006.
Abstract: Central European encephalitis is the most common arthropod-borne virus disease in the Czech Republic, with the mean annual incidence of 6 cases per 100 000 population. However, seven less known arboviruses (Flavivirus West Nile, Bunyavirus Tahyna, Bunyavirus Batai, Bunyavirus Sedlec, Bunyavirus Lednice, Orbivirus Tribee, Uukuvirus Uukuniemi) also circulate in this country, of which West Nile, Tahyna, Tribec and possibly Batai have been reported to cause human disease. Moreover, antibodies against two other pathogenic viruses found in Europe, i.e. Alphavirus Sindbis and Coltivirus Eyach, have been detected in the Czech Republic. The aim of this study is to review briefly the less known arboviruses found in the Czech Republic with emphasis on the taxonomic status, identification of their hosts and vectors, and pathogenicity to humans. These arboviruses can cause febrile illness to aseptic (meningo)encephalitis of unclear etiology. The review points out the possible emergence of these neglected arboviruses in the foreseeable future and provides diagnostic guidance.
Abstract: A survey for antibodies to West Nile virus (WNV; genus ,Flavivirus) was carried out by plaque-re-duction neutralization microtesting in 78 horses, 20 domestic chickens, and 97 wild birds belonging to 10 species from different areas in Poland. Specific antibodies were detected in five juvenile (hatching-year) birds collected in 2006: three white storks (Ciconia ciconia) in a wildlife rehabilitation center (5.4% of all examined storks; the antibody titers in each bird were 1:320, 1:160, and 1:20), one free-living mute swan (Cygnus olor; the titer was 1:20), and one hooded crow (Corvus corone cornix; the titer 1:20) in a wildlife rehabilitation center; thus the overall seropositivity to WNV was 5.2% among all the birds sampled. These data do not rule out the presence of WNV activity in Poland with 100% certainty, but they indicate a significant trace that demands verification. In addition, one black-headed gull (Larus ridibundus) had neutralizing antibodies for the Usutu Flavivirus, the first case recorded in Poland.
Abstract: ABSTRACT: BACKGROUND: The incidence of tick-borne encephalitis showed a dramatic spike in several countries in Europe in 2006, a year that was unusually cold in winter but unusually warm and dry in summer and autumn. In this study we examine the possible causes of the sudden increase in disease: more abundant infected ticks and/or increased exposure due to human behaviour, both in response to the weather. METHODS: For eight countries across Europe, field data on tick abundance for 2005-2007, collected monthly from a total of 41 sites, were analysed in relation to total annual and seasonal TBE incidence and temperature and rainfall conditions. RESULTS: The weather in 2006-2007 was exceptional compared with the previous two decades, but neither the very cold start to 2006, nor the very hot period from summer 2006 to late spring 2007 had any consistent impact on tick abundance. Nor was the TBE spike in 2006 related to changes in tick abundance. Countries varied in the degree of TBE spike despite similar weather patterns, and also in the degree to which seasonal variation in TBE incidence matched seasonal tick activity. CONCLUSION: The data suggest that the TBE spike was not due to weather-induced variation in tick population dynamics. An alternative explanation, supported by qualitative reports and some data, involves human behavioural responses to weather favourable for outdoor recreational activities, including wild mushroom and berry harvest, differentially influenced by national cultural practices and economic constraints.
Abstract: Mean first arrival dates (FAD) of 45 migratory bird species recorded in Moravia (Czech Republic) in 109 spring seasons between 1881 and 2007 were correlated with winter North Atlantic Oscillation (NAO) index. The arrival occurred significantly earlier following high NAO winter index values in all short-distance migratory species (with a European or North African winter range) whereas in long-distance migrants (with sub-Saharan winter range). When the values of Pearson coefficient between NAO and FAD were correlated with the migration distance of all 45 bird spp., the correlation indicated that the distance was responsible for 68-72% of variation in the regression of birds' arrival on winter NAO index in central Europe.
Abstract: The number of mosquito-borne viruses ('moboviruses') occurring in Europe since the twentieth century now stands at ten; they belong to three families-Togaviridae (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Bunyaviridae (Batai, Tahyna, Snowshoe hare, Inkoo, Lednice). Several of them play a definite role in human or animal pathology (Sindbis, Chikungunya, Dengue, West Nile, Tahyna). Mobovirus outbreaks are strictly determined by the presence and/or import of particular competent vectors of the disease. Ecological variables affect moboviruses considerably; the main factors are population density of mosquito vectors and their vertebrate hosts, intense summer precipitations or floods, summer temperatures and drought, and presence of appropriate habitats, e.g., wetlands, small water pools, or intravillan sewage systems. A surveillance for moboviruses and the diseases they cause in Europe is recommendable, because the cases may often pass unnoticed or misdiagnosed not only in free-living vertebrates but also in domestic animals and even in humans.
Abstract: A serosurvey for mosquito-borne viruses was carried out in 93 wild boars (Sus scrofa), using a plaque-reduction neutralization microtest with Vero cells. The boars were sampled on 24 hunting grounds of the BÅeclav district (South Moravia) from 2000 to 2002. Specific antibodies to Flavivirus West Nile (WNV) were detected in 6 (6.5%) animals only at Lanžhot and Kostice, i.e. in the area of the "Soutok" game reserve where WNV was isolated in South Moravia from mosquitoes previously. However, the antibody titres were comparatively low (1:20-1:40). A substantially higher seroprevalence was revealed against Orthobunyavirus ŤahyÅa (TAHV): 18 (19.4%) wild boars were positive, and the titres ranged from 1:20 up to 1:640. Only one animal (1.1%) seroreacted with Orthobunyavirus Batai (Äalovo), at a low titre of 1:20. The sera were additionally examined by haemagglutination-inhibition test against Alphavirus Sindbis: two boars (2.2%) revealed antibodies, the titres were 1:20 and 1:80. The serosurvey indicates that the activity of mosquito-borne viruses in South Moravia has decreased compared with the past decades, but surveillance for these viruses is necessary.
Abstract: The hepatitis E virus (HEV), the causative agent of hepatitis E (HE), is a non-enveloped RNA virus. HEV is classified in the genus Hepevirus, the only member of the Hepeviridae family. HE is usually transmitted via the faecal-oral route with contaminated drinking water or water for industrial purposes. Spread of HE has been reported in developing countries of Asia, Africa, South and Central America. Human cases in countries with the sporadic occurrence of HE have been associated with travelling to countries with an increased risk of infection. But HE has been described also in people who did not travel to endemic countries. Further studies have suggested other routes of transmission and the zoonotic potential (pigs and deer as the potential source of human infection) of the virus.
Abstract: A total of 150 nymphal Ixodes ricinus (L., 1758) (Acari: Ixodidae) from the Czech Republic were examined for Anaplasma phagocytophilum (Foggie, 1949) Dumler et al., 2001 by PCR using EHR521/747 primers: 22 of 50 pools were positive (minimum prevalence, 14.7%). However, sequencing of the PCR products did not show complete homology with A. phagocytophilum (91%) while the closest relationship (95%) was found to "Candidatus Ehrlichia walkefii". The results indicate a need for care in interpretation of Anaplasma PCR results and for PCR optimization for detecting A. phagocytophilum in ticks.
Abstract: A systemic disease occurred in a wild population of the common vole Microtus arvalis in South Moravia (Czech Republic) during the years 1999-2003. Acute infections were characterized by edema of extremities, occasionally with colliquating abscesses, arthritis, lymphadenitis, perforations of the skin resulting from colliquated abscesses, orchitis, and peritoneal granulomas. From the clinical samples, small Gram-negative coccobacilli were isolated and identified as Ochrobactrum intermedium by API 20NE and colistin sensitivity profiles. However, subsequent rrs (16S rRNA) and recA (recombinase A) gene sequencing analysis of two isolates (CCM 4915=CAPM 6434; CCM 4916=CAPM 6435) identified them as Brucella sp. with sequence identities of 100% to other Brucella spp. Analysis of the omp2a/b genes confirmed the two isolates as Brucella. In AMOS polymerase chain reaction (PCR), a 2000-bp fragment was generated that was not seen in other brucellae. Experimental infection of outbred ICR mice with these isolates resulted in a mortality rate of 50%. Based on the results of the molecular investigations and the mortality observed in experimentally infected mice we conclude that the epizootic was caused by Brucella sp. and not by Ochrobactrum intermedium. The study demonstrates the limitations of commercial biochemical test systems in accurately differentiating among Ochrobactrum and Brucella.
Abstract: West Nile virus (WNV) is principally considered to be maintained in a mosquito-bird transmission cycle. Under experimental conditions, several other transmission routes have been observed, but the significance of these additional routes in nature is unknown. Here, we derive an expression for the basic reproduction number (R0) for WNV including all putative routes of transmission between birds and mosquitoes to gauge the relative importance of these routes for the establishment of WNV. Parameters were estimated from published experimental results. Sensitivity analysis reveals that R0 is sensitive to transmission between birds via close contact, but not to mosquito-to-mosquito transmission. In seasons or in areas where the mosquito-to-bird ratio is low, bird-to-bird transmission may be crucial in determining whether WNV can establish or not. We explain the use of R0 as a flexible tool to measure the risk of establishment of vector-borne diseases.
Abstract: Status and population trends of sand martins (Riparia riparia) were studied over a fourteen year period in the Czech Republic. In sum, 438 occupied localites were found. The population fell off 83.2 % between years 1993 and 2005, which was unexpected as the population was stable or slightly increasing for years before. Number of holes per locality triplicated from 130 in 1992 to 314 in 2005 as a result of change of quarrying methods. In all but two years more colonies were lost than were formed. Among the localities preferred were sandpits (63.6 %), gravelsandpits (5.3 %), slopes and claypits (3.7 % each). Only 3.0 % of colonies were in riverbanks. Average altitude of colonies was 313.5 m a.s.l. with maxima around 650 m a.s.l. Negative influence of water precipitation during the breeding season and positive effects of precipitation in Subsaharan regions are described.
Abstract: We report West Nile virus infection of the central nervous system in a 69-year-old man, residing in North Moravia (Czech Republic), who visited the USA from 6 July to 31 August 2002. He developed fever with fatigue at the end of his US stay, and was hospitalized in Ostrava after his return on 3 September with fever (up to 39.5 degrees Celsius), fatigue, anorexia, moderate laryngotracheitis, dizziness, insomnia, blurred speech, and a marked bradypsychism. EEG demonstrated a slow bifrontal theta-delta activity, and CT of the brain a slight hydrocephalus. A significant increase of antibodies neutralizing West Nile virus was detected between the first (1:16) and second (1:256) blood serum sample. The patient recovered gradually and was released from hospital on 16 September. This is the first recorded human case of West Nile fever (WNF) imported to the Czech Republic. Nine similar cases of WNF import from the USA have already been reported in other European countries - France, Denmark, the Netherlands, and Germany.
Abstract: Highly pathogenic avian influenza (HPAI) H5N1 expanded considerably during 2005 and early 2006 in both avian host species and geographic distribution. Domestic waterfowl and migratory birds are reservoirs, but lethality of this subtype appeared to initially limit migrant effectiveness as introductory hosts. This situation may have changed, as HPAI H5N1 has recently expanded across Eurasia and into Europe and Africa. Birds could introduce HPAI H5N1 to the Western Hemisphere through migration, vagrancy, and importation by people. Vagrants and migratory birds are not likely interhemispheric introductory hosts; import of infected domestic or pet birds is more probable. If reassortment or mutation were to produce a virus adapted for rapid transmission among humans, birds would be unlikely introductory hosts because of differences in viral transmission mechanisms among major host groups (i.e., gastrointestinal for birds, respiratory for humans). Another possible result of reassortment would be a less lethal form of avian influenza, more readily spread by birds.
Abstract: A total of 691 Ixodes ricinus (22 male, 39 female, 501 nymphs and 129 larvae), the tick vector of Lyme borreliosis, were collected by flagging from vegetation in 11 areas at altitudes between 789 m and 1350 m above sea level in mixed woodland with pasture land (cattle) in the province of Styria in Austria. The ticks were individually examined for presence of Borrelia burgdorferi sensu lato by dark-field microscopy and 107 of them by real-time PCR. Attempts to cultivate borreliae were made in BSK-H medium. The overall positivity rate of all collected ticks (excepting larvae) was 10.9%: 9.1% in males, 17.9% in females and 10.4% in nymphs. The 129 larvae examined showed no presence of B. burgdorferi s.l. The mean infection rate of I. ricinus collected at the highest altitude in this study, Gaberl at 1350 m a.s.l.--and at the same time the highest one reported in Europe--was 6.4%: 1/9 males, 2/18 females and 6/114 (5.3%) nymphs were positive. Culture attempts were positive in 12 cases and species identification showed eight isolates were B. afzelii and four B. garinii. Three additional positive results found by PCR (negative by culture) were identified twice as B. afzelii and once as B. garinii. This study shows that the risk of acquiring Lyme borreliosis in habitats at higher altitudes is limited, because of the lower density of I. ricinus and its lesser infection rate than at lower altitudes in central Europe, but nevertheless the risk does exist.
Abstract: Questing Ixodes ricinus L. (Acari: Ixodidae) ticks were collected on a forest trail that had been completely cleared of shrubs and ground vegetation in winter 2002 and on a nearby control uncleared forest transect in South Moravia (Czech Republic). Samples were collected each May in 2003, 2004 and 2005. Nymphal ticks were 3.4 times, 1.9 times and 1.2 times less frequent on cleared forest than on uncleared forest trails in the three respective years, whereas adult tick abundance was 27.2 times, 4.0 times and 2.2 times lower, respectively. The ticks were examined for borreliae by dark-field microscopy: prevalence of nymphal ticks infected with Borrelia burgdorferi sensu lato (12.6% to 20.0%) did not differ significantly between the cleared and uncleared trail during the 3 years. In conclusion, the habitat modification appeared to result in a decreased abundance of I. ricinus as well as a reduced frequency of infected ticks (and thus indirectly a lower potential risk of Lyme borreliosis), which lasted, however, for only 2 years. Eight cultures of borreliae isolated from the ticks were all identified as the 'ornithophilic' genomic species Borrelia garinii, possibly indicating a greater role of forest birds than that of forest rodents as the hosts of immature I. ricinus in the tick (and borrelial) colonization of the cleared part of the forest.
Abstract: A flavivirus (strain 97-103) was isolated from Culex pipens mosquitoes in 1997 following floods in South Moravia, Czech Republic. The strain exhibited close antigenic relationship to West Nile virus (WNV) prototype strain Eg-101 in a cross-neutralization test. In this study, mouse pathogenicity characteristics and the complete nucleotide and putative amino acid sequences of isolate 97-103, named Rabensburg virus (RabV) after a nearby Austrian city, were determined. RabV shares only 75%-77% nucleotide identity and 89%-90% amino acid identity with representative strains of WNV lineages 1 and 2. Another RabV strain (99-222) was isolated in the same location 2 years later; it showed >99% nucleotide identity to strain 97-103. Phylogenetic analyses of RabV, WNV strains, and other members of the Japanese encephalitis virus (JEV) complex clearly demonstrated that RabV is either a new (third) lineage of WNV or a novel flavivirus of the JEV group.
Abstract: A total of 350 nymphs of the common tick Ixodes ricinus (Linnaeus, 1758) were collected in an endemic focus of Lyme borreliosis (South Moravia, Czech Republic) and examined for the presence of the protozoan Babesia microti (França, 1909) by polymerase chain reaction (PCR), using primers specific for the B. microti gene encoding small subunit rRNA. The assay revealed five positive pools (out of 70 pools examined); the corresponding prevalence rate was about 1.5%. Sequence analysis of the PCR products confirmed their 100% homology with that of B. microti. The study represents the first evidence of B. microti in ixodid ticks in the Czech Republic.
Abstract: The presence of adiaspores of the fungal genus Emmonsia was examined in the lungs of 85 mole rats representing 3 subterranean genera: blind mole rats (Spalax galili and S. golani) from Israel, Ansell's mole-rats (Cryptomys anselli) from Zambia, and silvery mole-rats (Heliophobius argenteocinereus) from Malawi and Zambia. Emmonsiosis was found in 28% of the blind mole rats, 100% of the Ansell's mole-rats, but in none of the silvery mole-rats. Infection in African mole-rats was caused by Emmonsia parva, and infection in Israeli blind mole rats was caused by E. parva and E. crescens. The study indicates that the perennial burrow system of the Ansell's mole-rat forms an appropriate microhabitat for the saprophytic growth of E. parva in Lusaka region, Zambia. We suggest that factors contributing to the striking difference in prevalence of emmonsiosis between the two African mole-rat genera (Cryptomys, Heliophobius) may be their differing burrow types, burrow longevity, and social lives.
Abstract: Longitudinal correlation between the North Atlantic Oscillation large-scale weather system (NAO) and the annual incidence rate of 14 viral, bacterial and protozoan national notifiable human diseases in the Czech Republic was examined. In simple correlation, cases of salmonellosis, erysipelas, infectious mononucleosis and toxoplasmosis were positively correlated with the winter NAO index, while hepatitis A and shigellosis were negatively correlated, and the other diseases tested (rubella, mumps, chickenpox, tick-borne encephalitis, Lyme borreliosis, leptospirosis, tularemia and scarlet fever) were uncorrelated with NAO. However, 8 of the 14 diseases also revealed a significant time trend, either increasing (infectious mononucleosis, salmonellosis, erysipelas, toxoplasmosis) or decreasing (hepatitis A, scarlet fever, leptospirosis, shigellosis) during the period. When the effect of NAO on incidence of the diseases was then controlled for calendar year using partial correlation analysis and detrended regression, only toxoplasmosis and infectious mononucleosis were found significantly positively correlated with the NAO when the index was lagged 1 or 2 years, and leptospirosis was correlated negatively with a lag of 2 years. Large-scale weather changes as described by NAO therefore do not seem to be a crucial factor in the fluctuation of annual incidence rate of the majority of tested infectious diseases in the Czech Republic, while other factors, especially social and public health circumstances, are obviously more important.
Abstract: Specimens from residents (N = 497) of an area affected by the 2002 flood were examined serologically for mosquitoborne viruses. Antibodies were detected against Tahyna (16%), Sindbis (1%), and Batai (0.2%) viruses, but not West Nile virus. An examination of paired serum samples showed 1 Tahyna bunyavirus (California group) infection.
Abstract: An episode of mortality in waterbirds occurred on a sedimentation reservoir with effluents from the sugar beet processing plant at Hrušovany n.J. (South Moravia, Czech Republic) in summer 2003: tens of black-headed gulls (Larus ridibundus), several lapwings (Vanellus vanellus), one little ringed plover (Charadrius dubius), one ruff (Philomachus pugnax), one wood sandpiper (Tringa glareola), and two avocets (Recurvirostra avosetta) died. One of the two avocets and the plover, local breeders, were examined and found positive for Clostridium botulinum type C toxin by toxin-neutralization test. Avocet is classified as a rare bird species according to Red Book data and protected as critically endangered in the Czech Republic. Avian botulism can occasionally bring severe damage to waterbird fauna and its protection.
Notes: PDF available on request from the author: zhubalek@brno.cas.cz
Abstract: In 1997 to 1999, spring arrival of migrants was recorded by 23 ornithologists in three areas of the South-Moravian region (Czechland) that were situated at altitudes of 150-200 m (N, BÅeclav area), 200-400 m (P, Brno and surroundings), and 500-750 m a.s.l. (V, ŽÄár n.S. area). The south-north distances among the three areas are: NâP 50-60 km, PâV 50-80 km, NâV 100-140 km. The timing of arrival in most species was gradual from the low-situated (southern, N) area to the elevated (northern, V) area. In 34 spp. analysed in detail, the first individuals were recorded in area N on average 3.9 days earlier than in area P, while 4.7 days earlier in area P than in area V; the mean difference between the areas N and V was 8.2 days. The whole region was occupied by 18 bird species (ârapid migrantsâ) within 10 days, while the interval was longer in 12 spp. The arrival was accelerated from the beginning to early April in all three years in correlation with the higher than normal air temperature, whereas arrival of the late-arriving spp. approximately corresponded to normal dates.
Abstract: Long-term spring arrival dates of 37 migratory bird species as recorded in Moravia (Czech Republic) during 103 years between 1881 and 2001 were evaluated for pairwise correlation (i.e. co-fluctuation in migratory timing) between avian species. Cluster analysis of the correlation matrix revealed a number of clusters (called 'migrons') of co-fluctuating migratory bird species. All short-distance migrants with the European (Mediterranean) winter range clustered together in migron A (Alauda arvensis, Motacilla alba, Vanellus vanellus, Sturnus vulgaris, Corvus frugilegus, Columba palumbus, Phoenicurus ochruros, Phylloscopus collybita, Remiz pendulinus, Erithacus rubecula, Turdus philomelos, Larus ridibundus, Serinus serinus, Sylvia atricapilla), while six other, smaller clusters were formed exclusively of long-distance migrants having an African (sub-Saharan) winter range: (B) Cuculus canorus, Streptopelia turtur; (C) Hirundo rustica, Jynx torquilla, Luscinia megarhynchos, Apus apus, Sylvia curruca; (D) Acrocephalus schoenobaenus, Riparia riparia, Upupa epops; (E) Anthus trivialis, Delichon urbica, Motacilla flava, Hippolais icterina; (F) Ciconia ciconia, Phoenicurus phoenicurus, Ficedula albicollis, Acrocephalus arundinaceus, Lanius collurio; (G) Oriolus oriolus, Muscicapa striata, Locustella fluviatilis, Coturnix coturnix. Results of the co-migration analysis pose interesting questions about possible varying underlying mechanisms of the migration timing in different migrons of birds.
Abstract: In the Central-Bohemian area affected by the flood of 2002, 497 residents were screened for antibodies against the mosquito-borne viruses Tahyna (TAHV), West Nile (WNV), Sindbis (SINV) and Batai (BATV; syn. Calovo) using the haemagglutination-inhibition (HIT) and plaque-reduction neutralization (PRNT) tests. Blood samples were collected in September 2002 when the mosquito populations showed the maximum density following the flood. Antibodies against TAHV (16.5% persons in PRNT, 14.9% in HIT), SINV (1.4% in HIT) and BATV (1.4% in HIT, 0.2% in PRNT) were detected. Although 6.8% and 1.2% of the subjects tested reactive with WNV in HIT and PRNT, respectively, the results were interpreted as cross-reactivity with tick-borne encephalitis virus. The seroprevalence of TAHV (both in PRNT and HIT) showed no association with gender (15.8% of males, 16.9% of females), increased with age (1.4% of persons younger than 20 years, 11.2% of persons aged between 20 and 50 years, and 26.2% of persons older than 50 years were positive), and correlated with the mosquito peri-residential challenge (5.0% residents seropositive in a mosquito-free control zone D--mostly Prague, 14.7% in a mild-risk zone C, 20.5% in a moderate-risk zone B, and 28.0% in the most heavily mosquito-infested risk zone A). The highest TAHV seropositivity rate (> 25%) was found amongst the inhabitants of the villages ObrÃstvÃ, Kozly, Tuhan, Chrást, ChlumÃn and HostÃn. Paired blood samples were obtained from 150 of the persons at a 6-month interval: an infection episode with TAHV during or after the flood was clearly evidenced in one person living in ObrÃstvÃ, and less convincing findings of recent TAHV infections were found in other three residents of ChlumÃn and ObrÃstvà (seroconversion and/or significant antibody titres increase detected in HIT only). This serosurvey indicated the existence of an active natural focus of Valtice fever (TAHV infection) stretched along the river Labe nearby Neratovice (ObrÃstvÃ, ChlumÃn, Tuhan; Kozly, Tisice, Chrást), and a low TAHV activity area along the lower reaches of the river Vltava between Zloncice and Bukol/Zálezlice. An increased population density of mosquitoes after the flood may have boosted the incidence of mosquito-borne virus diseases, particularly Valtice fever, in Central Bohemia. An optimum prophylactic strategy to control these diseases would be epidemiological surveillance (including monitoring of both the density of mosquitoes and their rate of infection with viruses in natural foci) on the basis of which antiepidemic measures such as integrated mosquito control can be taken.
Abstract: The North Atlantic Oscillation (NAO) has been used as a simple approximate descriptor of the global weather fluctuation over Europe. Spring arrival dates of 37 migratory bird species (summer visitors) recorded in Moravia, Czech Republic during 103 years between 1881 and 2001 were correlated with the seasonal NAO index. Bird arrivals occurred significantly earlier following positive winter/spring NAO values (causing a warmer spring than normal in Central Europe) in all short-distance migrants with a European (Mediterranean) winter range (Alauda arvensis, Anser anser, Columba palumbus, Larus ridibundus, Phoenicurus ochruros, Phylloscopus collybita, Remiz pendulinus, Saxicola torquata, Serinus serinus, Sturnus vulgaris, Turdus philomelos, Vanellus vanellus). On the other hand, the timing of arrival did not correlate significantly with seasonal NAO in long-distance migrants having largely an African (sub-Saharan) winter range (Acrocephalus schoenobaenus, Anthus trivialis, Apus apus, Ciconia ciconia, Cuculus canorus, Ficedula albicollis, Hippolais icterina, Hirundo rustica, Jynx torquilla, Lanius collurio, Luscinia megarhynchos, Muscicapa striata, Oriolus oriolus, Phylloscopus sibilatrix, Riparia riparia, Streptopelia turtur, Sylvia atricapilla, S. curruca, Upupa epops). The prevailing positive phase of winter/spring NAO conditions observed in Europe at the end of the 20th century has obviously determined the trend of an earlier than normal arrival of short-distance migratory species.
Abstract: The potential for transport and dissemination of certain pathogenic microorganisms by migratory birds is of concern. Migratory birds might be involved in dispersal of microorganisms as their biological carriers, mechanical carriers, or as carriers of infected hematophagous ecto-parasites (e.g., ixodid ticks). Many species of microorganisms pathogenic to homeothermic vertebrates including humans have been associated with free-living migratory birds. Migratory birds of diverse species can play significant roles in the ecology and circulation of some arboviruses (e.g., eastern and western equine encephalomyelitis and Sindbis alphaviruses, West Nile and St. Louis encephalitis flaviviruses), influenza A virus, Newcastle disease virus, duck plague herpes-virus, Chlamydophila psittaci, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Campylobacter jejuni, Salmonella enterica, Pasteurella multocida, Mycobacterium avium, Candida spp., and avian hematozoans. The efficiency of dispersal of pathogenic microorganisms depends on a wide variety of biotic and abiotic factors affecting the survival of the agent in, or disappearance from, a habitat or ecosystem in a new geographic area.
Abstract: A total of 298 Ixodes ricinus (L.) ticks (Acari: Ixodidae) feeding on humans in the Czech Republic were tested for borreliae (Borrelia burgdorferi sensu lato) by darkfield microscopy between 1997 and 2003. A majority (68%) of the supplied I. ricinus ticks were nymphs, 25% were females and 7% were larvae. Overall, 20% of 74 examined females and 9% of 203 examined nymphs (but none of 21 examined larvae) were infected with borreliae. The proportion of ticks with a high infection load (>100 spirochetes) was 4% in females and 2% in nymphal I. ricinus. During the year, the highest numbers and proportions of infected nymphal and female ticks were taken from humans in June. Detection of borreliae in the ticks feeding on humans might be helpful in the prophylaxis of Lyme borreliosis.
Abstract: West Nile fever (WNF), caused by mosquito-borne West Nile flavivirus (WNV), is one of the emerging infectious diseases. WNV is geographically the most widespread member of the family Flaviviridae, occuring in Africa, Eurasia, Australia (Kunjin subtype) and, since 1999, surprisingly also in North America. In Europe, WNF epidemics were described in southern France and Russia in the 1960's, then in Portugal, Ukraine and Belarus. Two extensive epidemics occurred in southern Romania and Russia recently (1996-2000), each of them with nearly 1000 patients. A low activity of WNV was also detected in the Czech Republic in 1997 (five patients). A review of clinical symptoms (the most severe being encephalitis), laboratory diagnosis, prevention and epidemiological surveillance of WNF is given. Susceptibility of vertebrates other than humans to WNV infection, ecology of the virus (arthropod vectors, vertebrate hosts, natural cycles) and potential impact of climate changes and other factors on the distribution of WNV are also described briefly in the review.
Abstract: Long-term spring phenological instants of 57 migratory bird species, i.e. arrival in summer visitors and departure in winter visitors, were recorded in South Moravia (Czech Republic) from 1952 through 2001 and evaluated for annual correspondence with the North Atlantic Oscillation (NAO) weather system. The migration instants occurred significantly earlier following positive winter/spring NAO index values (causing periods warmer than normal in Europe) in a number of short-distance migrants with a European winter range (e.g., Alauda arvensis, Columba palumbus, Corvus frugilegus, Motacilla alba, Phoenicurus ochruros, Phylloscopus collybita, Serinus serinus, Sturnus vulgaris, Vanellus vanellus), whereas they did not correlate with NAO in most long-distance migrants having a sub-Saharan winter range (e.g., Acrocephalus spp., Anthus trivialis, Apus apus, Cuculus canorus, Delichon urbica, Ficedula albicollis, Hippolais icterina, Hirundo rustica, Jynx torquilla, Lanius collurio, Locustella spp., Muscicapa striata, Oriolus oriolus, Phylloscopus sibilatrix, Riparia riparia, Streptopelia turtur, Sylvia spp.). The winter/spring (especially February and March) NAO conditions thus affect the migration timing of short-distance migrants that winter in western or southern Europe, and could explain their earlier than normal arrival that had been observed in Europe since the 1980s.
Abstract: Host-seeking ixodid ticks were sampled in a floodplain forest ecosystem along the lower reaches of the Thaya (Dyje) river in South Moravia (Czech Republic) and Lower Austria during the period 1989-2002. The ticks were examined by dark-field microscopy for borreliae (Borrelia burgdorferi sensu lato, the agent of Lyme borreliosis), and attempts were made to culture the spirochetes in BSK-H medium from preparations containing their high numbers. Isolated borreliae were identified by PCR-RFLP analysis using probes directed against ribosomal spacer genes. A total of 797 nymphal and 719 adult (391 female, 328 male) Ixodes ricinus were examined: 16.2% of nymphs, 28.6% of females and 29.0% of males were positive. Dermacentor reticulatus (70 females, 30 males) and Haemaphysalis concinna (12 nymphs, 8 females, 2 males) were negative for spirochetes. The overall prevalence rate of borreliae in I. ricinus from the floodplain forest is slightly higher than the mean European data (i.e., 14% for nymphs, 21% for adults). The difference in infection rate between nymphal and adult ticks was significant, including the proportion of heavily infected (with > 100 borreliae) nymphs (2.1%) vs. adults (7.6%). Prevalence of borreliae in I. ricinus showed a significant decrease during autumn in this ecosystem. Three strains of spirochetes, all of the Borrelia afzelii genomic group, were isolated from female I. ricinus. Moreover, Trypanosoma/Crithidia sp. protozoa and Dipetalonema rugosicauda nematodes were detected in 0.4% and 1.0%, respectively, of all I. ricinus.
Abstract: The cryoprotective additives (CPAs) used in the frozen storage of microorganisms (viruses, bacteria, fungi, algae, and protozoa) include a variety of simple and more complex chemical compounds, but only a few of them have been used widely and with satisfactory results: these include dimethylsulfoxide (Me2SO), glycerol, blood serum or serum albumin, skimmed milk, peptone, yeast extract, saccharose, glucose, methanol, polyvinylpyrrolidone (PVP), sorbitol, and malt extract. Pairwise comparisons of the cryoprotective activity of the more common CPAs used in cryomicrobiology, based on published experimental reports, indicate that the most successful CPAs have been Me2SO, methanol, ethylene glycol, propylene glycol, and serum or serum albumin, while glycerol, polyethylene glycol, PVP, and sucrose are less successful, and other sugars, dextran, hydroxyethyl starch, sorbitol, and milk are the least effective. However, diols (as well as some other CPAs) are toxic for many microbes. Me2SO might be regarded as the most universally useful CPA, although certain other CPAs can sometimes yield better recoveries with particular organisms. The best CPA, or combination of CPAs, and the optimum concentration for a particular cryosensitive microorganism has to be determined empirically. This review aims to provide a summary of the main experimental findings with a wide range of additives and organisms. A brief discussion of mechanisms of CPA action is also included.
Abstract: Six tests for the detection of West Nile virus (WNV) antibodies in the serum of experimentally infected chickens were compared. The tests included the hemagglutination-inhibition test (HIT), immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) with WNV-infected mouse brain antigen, immunoglobulin G (IgG) indirect ELISA with tickborne encephalitis viral antigen, the microtitre virus neutralization test, the standard plaque reduction neutralization test (PRNT), and the microtitre PRNT (micro-PRNT). Thirty adult chickens, intravenously and intramuscularly inoculated with 10(7) plaque-forming units (PFU) of WNV strain Egypt 101, were bled and given a booster of 10(7) PFU at 7,15, and 21 d postinoculation; the final blood collection was on day 28. Although the micro-PRNT is capable of detecting the highest antibody titres during both early and late infection, because of the technical complexity and time requirements of this test a combination of IgM and IgG ELISAs is recommended for serologic screening. Serum samples that give positive results in the ELISAs can then be tested by the micro-PRNT to determine the specificity of antibodies to WNV.
Abstract: Human babesiosis is a zoonotic tick-borne protozoan infection caused by several species of the genus Babesia. It is a rare disease in Europe. Until recently, 31 cases have been described. Most of them were severe infections caused by a cattle parasite Babesia divergens in splenectomized patients. In contrast, a rodent species Babesia microti was responsible for hundreds of cases of human babesiosis in the U.S.A. In this report we describe the first case of human babesiosis in the Czech Republic. To our knowledge, it is also the first case of symptomatic B. microti infection imported to Europe from the United States.
Abstract: The proportion of motile spirochetes decreased more slowly with the salivary gland extracts (SGE) of Ixodes ricinus than in corresponding control (C) since day 9 post inoculation (p.i.). On the other hand, the percentage of spirochetal motility decreased more rapidly with SGE and midgut extract (MGE) from D. reticulatus than in C since day 9 p.i. With I. ricinus, the concentration of motile spirochetes increased significantly from days 2 to 11 (p.i.) with both SGE and MGE compared to C. In addition, the growth of spirochetes was enhanced to a greater degree with SGE than with MGE on days 4, 7 and 9 p.i. With D. reticulatus, a significant increase in concentration of motile spirochetes was only detected with SGE (compared to C) on day 5 p.i., while a marked decrease in concentration of motile spirochetes was observed on day 9 p.i. with MGE, and on day 12 p.i. with both extracts compared to C. Moreover, many spirochetes grown in the presence of D. reticulatus MGE were morphologically changed (compared to C and SGE) by 9 days p.i.; the cells were damaged (e.g., less discernible walls), shorter, and with a lower number of coils. The effect of SGE and MGE on the growth of B. garinii spirochetes in vitro thus differed between the two tick species tested. While extracts derived from I. ricinus (a competent vector for Lyme borreliosis) stimulated growth significantly, extracts from D. reticulatus (a non-competent species) did not affect the growth of borreliae markedly, or even inhibited their growth on days 9 (MGE) and 12 p.i. (MGE and SGE). Our results therefore indicate that the tick compartment extracts surprisingly need not be inhibitory for pathogen survival in the body of even non-competent tick species like D. reticulatus in a short-term exposure.
Abstract: Ixodid ticks were monitored in a temperate deciduous broad-leaved forest in South Moravia (Czech Republic). Relative abundance of the ticks collected before noon (10.00-12.00 h) was compared to several weather variables (air and soil temperatures, relative humidity, precipitation, wind speed, and derived values) using the Pearson correlation coefficient. The tick numbers were found to be most closely related to the amplitude of the soil (-5 cm) temperature between 07 h and 14 h (TSamp, in Ixodes ricinus), and the soil temperature (TS) at noon (in Haemaphysalis concinna) or in the morning (Dermacentor reticulatus). While a growing amplitude in TSamp caused an increased host-seeking activity of I. ricinus and H. concinna, it suppressed the activity of D. reticulatus, a tick species mainly occurring in colder seasons of the year in Central Europe. The air temperature (TA) and relative humidity (RH) were also closely related to the tick activity, whereas rainfall and wind speed remained largely uncorrelated with the activity of the three tick species. Multiple linear regression on several variables (TSamp, TA, TS, TA-TS, RH) explained 48% of the variance in I. ricinus, 47% in H. concinna, and 38% in D. reticulatus. Predictive two-variable regression models of relative abundance in host-seeking ticks were based on morning temperature (TA or TS) and morning RH as the most important environmental factors: they explained 32% (I. ricinus), 39% (H. concinna), and 35% (D. reticulatus) of the variance.
Abstract: Avian mortality and encephalomyelitis in equines are considered good indicators for West Nile virus (WNV) activity. We retrospectively tested 385 horse sera for WNV antibodies and looked for WNV nucleic acid and/or WNV antigen in paraffin embedded tissues from 12 horses with aetiologically unresolved encephalomyelitis and 102 free-living birds of different species which had been found dead. With the exception of four horses originating from eastern European countries investigated on the occasion of transit through Austria, all horse sera were negative. Nested RT-PCR of the horse tissues yielded no amplification of WNV-RNA. Also, all bird samples, examined by immunohistochemistry, in situ hybridization and nested RT-PCR were negative for WNV. These results indicate that currently WNV cannot be considered a significant pathogen in Austria.
Abstract: West Nile virus was first recorded in the New World during August 1999 in New York City. Aetiology of the disease in the Old World indicated birds as the likely introductory and amplifying hosts with ornithophilous mosquitoes, e.g. Culex pipiens, as the principal vectors. Speculation regarding likely agents for movement of the virus in its new environment focused on migratory birds, but evidence to date is equivocal. While spread of the disease has been fairly rapid, at a rate of roughly 70 km a month, it has not shown the kind of long-distance, leap frog movements one might expect if transient birds were the principal introductory hosts. Furthermore, movement of the disease has not been focused southward, but shows a radiating pattern with detection sites located in all directions from New York where terrestrial habitat was available. In addition, tests among potential New World, avian hosts have revealed prolonged viraemia (up to 5 days) only in the relatively non-migratory House Sparrow (Passer domesticus). Dispersal movements by this species could account for the observed pattern of West Nile virus spread in the Western Hemisphere to date. Regardless of whether avian migration, dispersal, or some other agent is responsible, West Nile virus should reach the New World tropics in another 1-2 years, at which time a vast number of new potential introductory and amplifying avian hosts would be exposed to the disease and mosquito vectors would be available throughout most of the year, likely causing serious, long-term threats to human health and vulnerable avian populations in the region.
Abstract: Host-seeking Ixodes ricinus (L.) (Acari: Ixodidae) were monitored for borreliae (Borrelia burgdorferi s.l.) using dark-field microscopy in South Moravia (Czech Republic) each May from 1991 to 2001 (150 nymphs, 100 females and 100 males each year). This survey revealed a mean annual percentage of infected ticks of 16.8% (range, 11.7-24.2) in nymphs, 24.9% (range, 16.5-33.6) in females and 26.1% (range, 17.1-37.3) in males. Annual incidence of Lyme borreliosis in humans of the area in the same period (range, 8.7-41.7 per 100,000) correlated significantly with the frequency (number of ticks per flag per hour) of nymphs infected with >50 borreliae or all nymphal ticks, but not with the frequency of females, infected females or the infection rate (% of ticks infected) of either nymphal or female ticks. A prediction of the annual incidence of Lyme borreliosis, based on the frequency of heavily infected or all nymphal I. ricinus ticks, is feasible. The infection rate in I. ricinus correlated significantly with the North Atlantic Oscillation winter index of the last year (in nymphs) or of the year before last (in adults).
Abstract: According to the source of infection, any communicable human disease could be classified as either anthroponosis (when the source is an infectious man), zoonosis (the source is an infectious animal) or sapronosis (the source is an abiotic substrate - e.g. soil, water, decaying plants, animal excreta or carcasses, etc.). The source of infection means its reservoir or, in ecological terms, the habitat where the etiologic agent of disease normally thrives and replicates. Diseases of the last category - sapronoses - have remained a largely neglected, unrecognized and underdiagnosed group of human infections. We should expect an increase in the number of these infections including possible appearance of some new diseases that could bear the label of 'emerging sapronoses', in addition to emerging anthroponoses and zoonoses.
Abstract: We examined by dark field microscopy 195 of 209 Ixodes ricinus ticks that were removed from humans in the Czech Republic (mainly in southern Moravia) during 1997-2001. The majority of the ticks were nymphs (62%), 31% were females and 7% larvae; 10.7% of the nymphs, 20.3% of the females but no larvae were found to be infected with B. burgdorferi s.l., the agent of Lyme borreliosis (LB). This observation supports for Central Europe the conclusions drawn from North America about nymphal ixodid ticks as the most important vector stage in transmission of LB. In the course of the year, we found the highest proportion of infected nymphal and female ticks taken off humans in June. The detection of borreliae in a human-biting tick is an important step that enables the general practitioner to prescribe prophylactic antimicrobial treatment and to reduce significantly the risk of Lyme disease in the patient.
Abstract: Sera of 204 wild boars (Sus scrofa), shot by hunters in the South-Moravian district of BÅeclav during 1993-2001, were tested by microagglutination reaction using safranin-stained antigens of Francisella tularensis and Brucella abortus: 10.8% and 8.7% seroreactors, respectively, were detected. The highest (17%) prevalence of tularaemia antibodies was found in wild boars during 1993-1994 at the beginning of the widespread outbreak of tularaemia in South Moravia that started in 1994, a nonsignificantly lower (13%) seroprevalence in 1995-1996 during the continuing epizootic, whereas it decreased markedly to 3% in the years 1997-2001 during the disappearance of the epizootic. Brucella antibodies were significantly most frequent (15%) in wild boars in the years 1995-1996. This Brucella seroreactivity has been attributed to B. suis biotype 2 (B. melitensis biovar Suis biotype 2 according to new nomenclature) infection, because B. abortus in both cattle and humans (Bang's disease) was eradicated in the former Czechoslovakia in 1964. The hare brucellosis (B. suis biotype 2) has occurred in the BÅeclav district in a number of natural foci revealing an increased activity since 1994.
Notes: PDF available on request from the author: zhubalek@brno.cas.cz
Abstract: According to the source of infection, any human disease could be classified as anthroponosis (when the source is an infectious man), zoonosis (the source is an infectious animal - domestic or wild), or sapronosis (the source is a non-living substrate or abiotic environment - e.g. soil, water, animal excreta, decaying plants etc.). The source of infection means its reservoir or, in ecological terms, the habitat where the etiologic agent of disease normally thrives and replicates. The sapronoses have remained a largely neglected, unrecognized and/or underdiagnosed group of human infectious diseases in medical microbiology. We should expect an increase in their number including possible appearance of some new diseases.
Notes: In Czech, with a summary in English. Related paper: Hubalek Z. (2003) Emerging human infectious diseases: anthroponoses, zoonoses, and sapronoses. Emerg. Infect. Dis. 9: 403-404
Abstract: Southern Moravia is one of the areas where there are long-term natural foci of tularaemia. In 1994 in the Breclav district an exacerbation of the disease was recorded in hares. During autumn hunts a positive reaction was recorded in 5.75% of examined hares. An elevated seropositivity persisted also in subsequent years and at the same time a higher human morbidity in the mentioned region. The objective of the investigation was to assess the causes and circumstances which led to the exacerbation of old known but in recent years quiescent foci in the mentioned region. To this end 350 small terrestrial mammals were examined. In one case it proved possible to detect the causal agent in Microtus arvalis. By examination of arthropods it proved possible to isolate 33 strains, i.e. 32 strains from ticks Dermacentor reticulatus and one strain from Ixodes ricinus. The results of the examination revealed that tularaemia as a classical infection with a natural focus may persist in areas where there are prerequisites for survival of the causal agent in the environment. One of the important factors of the environment are susceptible individuals (hares, small terrestrial mammals) as well as blood sucking arthropods as vectors of the disease.
Abstract: Activities of 19 enzymes were tested by the API ZYM system in 13 strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, B. valaisiana) grown in liquid BSK-H medium supplemented with rabbit serum. All strains produced acid phosphatase, esterase (C4), esterase-lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Nine strains also produced alkaline phosphatase, and three strains produced alpha-glucosidase. The API ZYM system probably cannot be used for differentiation between B. burgdorferi sensu lato genomospecies.
Abstract: I compared the clinical picture of WNF in 9 selected outbreaks/countries: New York City 1999 (NYC99, 60 cases); Israel 1953 (ISR53, 70 cases); South Russia 1999 (RUS99, 826 cases); West Ukraine 1985 (UKR85, 38 cases); Romania 1999 (ROM99, 393 cases); Czechland 1997 (CZE97, 5 cases); South France 1962 64 (FRA63, about 20 cases); Congo 1998 (CON98, 45 cases); South Africa 1974 (RSA74, hundreds of cases). 20 clinical symptoms were all scored 0, 1, 2 and 3 according to the frequency of their occurrence among WNF patients (absent, <20%, 20 50%, and >50% patients, respectively). The mean score (and the ranges in parentheses) during the 9 outbreaks were calculated for individual symptoms: fever 3.0 (3), headache 3.0 (3), muscle ache 2.7 (2 to 3), joint pain 2.1 (1 to 3), fatigue 2.6 (2 to 3), nausea/vomiting 2.4 (1 to 3), abdominal pain 0.4 (0 to 2), diarrhea 0.8 (0 to 3), sore throat 1.6 (0 to 3), cough 0.7 (0 to 2), lymphadenitis 1.6 (0 to 3), skin rash/flushed face 2.1 (1 to 3), conjunctivitis 0.7 (0 to 2), ocular pain 1.1 (0 to 3), hepatosplenomegaly 0.4 (0 to 1), myocarditis 0.3 (0 to 1), meningitis 1.8 (1 to 2), encephalitis 1.1 (0 to 2), anterior myelitis 0.25 (0 to 1), fatality 0.3 (0 to 1). The matrix of the 20 scored symptoms in 9 countries was used for calculation of the average Manhattan distance (i.e., arithmetic average of absolute differences in the scores over all symptoms) between pairs of countries, and these distance coefficients were then subjected to UPGMA cluster analysis, and resulting dendrogram showed the relationships in clinical symptoms of WNF patients among the 9 countries/outbreaks. Three clusters of related symptomatology pattern were revealed: (i) recent outbreaks in Romania, Russia, Congo and USA; (ii) Israel epidemic in the 1950's; (iii) older outbreaks in France, Ukraine and South Africa. The latter cluster has been joined by Czech WNF cases, but those were only few and therefore the clustering is less reliable. After inspection, the first cluster is characteristic by a comparatively low frequency of some 'classic' WNF clinical symptoms (sore throat, maculopapular rash, ocular pain), whereas by a relatively higher rate of encephalitis and fatalities. The overall impression of the symptomatology relationships is that the time period (year) could probably be a more important factor of the symptom patterns than the geographic situation of the WNF outbreak. This might indicate the role of WN virus strains differing in their pathogenicity along the time scale; it seems that more virulent strains have been involved in recent outbreaks.
Abstract: A bird census was carried out on a study plot along the lower reaches of the River Dyje in South Moravia (Czech Republic) comprising 200 visits distributed homogeneously between seasons from 1986 to 1998. A forest habitat preference index was suggested and estimated for each species as FHP = dF/(dF + dO), where dF is the density of a bird species in forest (woodland) habitats, and dO is the density of the species in all other habitats. Thirty-seven species with the mean annual FHP values higher than 0.50 were regarded arbitrarily as 'forest' birds preferring woodland over non-forest habitats (e.g., Aegithalos caudatus, Certhia brachydactyla, Coccothraustes coccothraustes, Columba oenas, Dendrocopos major, D. medius, D. minor, Dryocopus martius, Erithacus rubecula, Ficedula albicollis, Fringilla coelebs, Garrulus glandarius, Hippolais icterina, Muscicapa striata, Parus caeruleus, P. major, P. palustris, Phylloscopus collybita, P. sibilatrix, P. trochilus, Sitta europaea, Sylvia atricapilla, Troglodytes troglodytes, Turdus viscivorus), whereas 54 species with FHP <0.30 as largely 'forest-avoiding' birds. However, analysis by season revealed a marked heterogeneity in preference for forest among bird species, and even some 'non-forest' species with the mean annual FHP <0.30 preferred woodland in certain periods of the year, e.g. during the breeding (Phasianus colchicus, Gallinula chloropus, Fulica atra, Cuculus canorus, Jynx torquilla, Picus canus, Motacilla alba, Oriolus oriolus, Corvus corone, Sturnus vulgaris, Passer montanus, Carduelis chloris, Serinus serinus, Emberiza citrinella), post-breeding or migration (Nycticorax nycticorax, Anas platyrhynchos, Falco tinnunculus, Saxicola torquata, Lanius excubitor) or winter (Alcedo atthis, Turdus pilaris, Carduelis carduelis) seasons. It is therefore difficult to define some species unequivocally as either 'forest-avoiding' or 'forest' birds.
Abstract: Twenty-four measures of species diversity, richness and equitability were compared using both real species abundance data (bird censuses on a standard study plot) and simple simulation tests. Based on 20 criteria, as the most recommendable measures for the estimation of alpha diversity of species have been evaluated Fager's "number of moves per specimen", exponential Shannon's information (or H'), reciprocal Simpson's lambda, and species richness (number of species). However, the last index is only appropriate when the sample sizes are approximately equal, otherwise it can be misleading and Hurlbert's rarefaction method should be applied. The other tested measures, especially all equitability indices, have been shown to be objectionable and should not be regularly used for the measurement of species diversity.
Notes: PDF available on request from the author: zhubalek@brno.cas.cz
Abstract: West Nile virus, an Old World flavivirus related to St. Louis encephalitis virus, was first recorded in the New World during August 1999 in the borough of Queens, New York City. Through October 1999, 62 patients, 7 of whom died, had confirmed infections with the virus. Ornithophilic mosquitoes are the principal vectors of West Nile virus in the Old World, and birds of several species, chiefly migrants, appear to be the major introductory or amplifying hosts. If transovarial transmission or survival in overwintering mosquitoes were the principal means for its persistence, West Nile virus might not become established in the New World because of aggressive mosquito suppression campaigns conducted in the New York area. However, the pattern of outbreaks in southern Europe suggests that viremic migratory birds may also contribute to movement of the virus. If so, West Nile virus has the potential to cause outbreaks throughout both temperate and tropical regions of the Western Hemisphere.
Abstract: A review of West Nile virus (WNV) and the epidemiology of West Nile fever (WNF) in Europe is presented. European epidemics of WNF reveal some general features. They usually burst out with full strength in the first year, but few cases are observed in the consecutive 1 to 2 (exceptionally 3) years, whereas smaller epidemics or clusters of cases only last for one season. The outbreaks are associated with high populations of mosquitoes (especially Culex spp.) caused by flooding and subsequent dry and warm weather, or formation of suitable larval breeding habitats. Urban WNF outbreaks associated with Culex pipiens biotype molestus are dangerous. Natural (exoanthropic, sylvatic) foci of WNV characterized by the wild bird-ornithophilic mosquito cycle probably occur in many wetlands of climatically warm and some temperate parts of Europe; these foci remain silent but could activate under circumstances supporting an enhanced virus circulation due to appropriate abiotic (weather) and biotic (increased populations of vector mosquitoes and susceptible avian hosts) factors. It is very probable that WNV strains are transported between sub-Saharan Africa and Europe by migratory birds. The surveillance system for WNF should consist of four main components: (1) monitoring of mosquito populations and their infection rate; (2) wild vertebrate surveys; (3) sentinel birds (domestic ducks rather than chickens); and (4) monitoring of human disease. In the case of persisting high risk of WNF for humans and equids in certain enzootic areas, immunization against WNF should be considered. For that purpose a commercially available, cross-protective vaccine against Japanese encephalitis could be used.
Abstract: Seven virus isolates were obtained from 11,334 mosquitoes after the 1997 Morava River flooding in South Moravia (Czech Republic): 6 strains of Tahyna bunyavirus, California antigenic group (5 from Aedes vexans, 1 from Ae. cinereus), and 1 strain of West Nile flavivirus (WNV) from Culex pipiens. In 1999, one isolate of Tahyna virus from Ae. vexans and one isolate of WNV from Cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there in 1,179 overwintering mosquitoes (mostly Cx. pipiens) in March 2000. The infection rate of mosquitoes with arboviruses was significantly higher in 1997, the year of the flood and an enormously high population density of mosquitoes. Antibodies neutralizing WNV were detected in 13 of 619 (2.1%) hospitalized patients or persons seeking outpatient clinics of the area in 1997. Five of the seroreactors revealed clinical symptoms compatible with West Nile fever: in 2 of them (children), recent infection with WNV was confirmed by a significant increase of antibody titer between acute and convalescent serum samples.
Abstract: We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.
Abstract: Adiaspores of the fungus Emmonsia crescens were detected microscopically in the lung tissue of 13% of 10.081 small mammals belonging to 24 species examined in 14 areas of the Czech Republic between 1986 and 1997; 441/1.934 (23%) Clethrionomys glareolus, 1/6 (17%) Arvicola terrestris, 357/2.172 (16%) Apodemus flavicollis, 220/1.981 (11%). A sylvaticus, 23/265 (9%) A. microps, 11/81 (14%) Microtus subterraneus, 93/1.275 (7%) M. arvalis, 98/1.439 (7%) M. agrestis, 1/3 (33%) Ondatra zibethicus, 1/1 Cricetus cricetus, 1/20 (5%) Crocidura suaveolens, 2/40 (5%) Neomys fodiens, and 13/529 (2%) Sorex araneus were infected. Emmonsiosis was not recorded among the species of rodents that do not build their nests in the soil (Muscardinus avellanarius, Micromys minutus, Mus musculus, Rattus norvegicus). The overall prevalence of emmonsiosis was significantly higher in adult (19%) than in juvenile (7%) mammals, and in rodents (13%, and 20% in adults) than in insectivores (2%, and 4% in adults). The frequency of infected mammals also varied according to geographic area, altitude, habitat, and season.
Abstract: Birds were censused along the lower reaches of the River Dyje (South Moravia, Czechland) between 1986 and 1998, using the belt transect method. The transect length was 12,650 m, and 5 to 8 morning counts were made in each of the 36 ten-day periods of the year (203 visits in total). Species richness, diversity and equitability of avian communities culminated in the breeding season (May-June), whereas abundance was highest between July and Sept., and biomass peaked in September and mid-winter. Overall, 164 spp. were recorded: 6 spp. were dominant (Parus major, Passer montanus, Parus caeruleus, Carduelis carduelis, Emberiza citrinella, Sitta europaea) , 9 spp. subdominant (Anas platyrhynchos, Fringilla coelebs, Sturnus vulgaris, Hirundo rustica, Corvus frugilegus, Passer domesticus, Aegithalos caudatus, Carduelis spinus, Larus ridibundus) and 11 spp. recedent (Anser anser, Phylloscopus collybita, Erithacus rubecula, Serinus serinus, Parus palustris, Turdus merula, Coccothraustes coccothraustes, Sylvia atricapilla, Dendrocopos major, Riparia riparia, Turdus pilaris).A considerable seasonal variation of the dominance structure of communities was documented.
Abstract: In July 1997, devastating floods occurred after heavy rains in Moravia, Czech Republic. Mosquito populations increased abruptly in the flooded area thereafter. We carried out a surveillance for mosquito-borne virus infections in the Breclav area, South Moravia, including serosurveys of inhabitants. A total of 11,334 female mosquitoes in 117 pools (9,100 Aedes vexans, 917 A. cinereus, 11 A. cantans, 1,074 A. sticticus and 232 Culex pipiens pipiens) were examined by virological methods. Seven virus isolates were obtained: six of them Tahyna virus (Aedes vexans 5, A. cinereus 1), while one was West Nile (WN) virus (Culex p. pipiens--first isolation in the Czech Republic). Sera of 619 local inhabitants were examined in plaque-reduction neutralization test, and antibodies to Tahyna virus were detected in 333 (53.8%) and to WN virus in 13 (2.1%). In 72 individuals, paired sera were sampled: a significant increase of antibody titre was detected once against Tahyna virus (a subclinical infection) and 4 times against WN virus (two children had an illness compatible with WN fever). Of the nine remaining WN seroreactors, three other revealed clinical symptoms compatible with WN fever in summer 1997. The data indicate WN virus activity in the Breclav area, and describe the first cases of WN fever in Central Europe. The WN virus should not be underestimated as a potential agent of local epidemics even in the temperate climate of Central Europe. Environmental factors including human activities which enhance vector population densities (heavy rains followed by floods, irrigation, higher temperature) can produce an increased incidence of mosquito-borne diseases, including WN fever.
Abstract: During the years 1995-1996, a total of 1,743 overwintering Culex pipiens biotype molestus female mosquitoes were tested for the presence of spirochetes in several localities in South Moravia, Czech Republic.The spirochetes were observed in 5% of the mosquitoes investigated. One of the five isolated strains of spirochetes (BR-84) was identified as Borrelia afzelii. The potential role of mosquitoes in the ecology and epidemiology of Lyme disease (LD) borreliae should be further investigated.
Abstract: Seventy-one isolates of Borrelia burgdorferi sensu lato (B.b.s.l.) derived from Ixodes ricinus ticks (50 strains) and patients (21 strains) were characterised by PCR-RLFP analysis. In four cases the human isolates were obtained from the cerebrospinal fluid (CSF) of patients with clinical symptoms of neuroborreliosis and in 17 cases from skin biopsies of patients with dermatological manifestation of Lyme borreliosis. Ixodes ricinus isolates originated from 14 localities in three regions (Mur valley, eastern and western Styria) in Styria. Thirty six strains of B.b.s.l. were isolated from nymphal ticks, nine strains from female and five strains from male ticks. Species identification of human isolates revealed three B. garinii and one B. afzelii isolates in CSF. In the PCR-RFLP analysis of 17 skin specimens a pattern for B. afzelii was found in ten cases, while six could be identified as B. garinii and one as a mixed infection of B. afzelii and B. garinii. Genetic characterisation of tick isolates resulted in 24 strains of B. afzelii (48%), 11 strains of B. garinii (40%) and 5 strains of B. burgdorferi s.st. (10%); one isolate showed a mixed infection of B. afzelii and B. garinii. Our findings indicate that B. afzelii and B. garinii predominate over B. burgdorferi s.str. in Ixodes ricinus ticks from Styria, which is similar to findings in neighbouring countries. This also reflects the occurrence of different pathogenic Borrelia strains in human samples.
Abstract: West Nile virus causes sporadic cases and outbreaks of human and equine disease in Europe (western Mediterranean and southern Russia in 1962-64, Belarus and Ukraine in the 1970s and 1980s, Romania in 1996-97, Czechland in 1997, and Italy in 1998). Environmental factors, including human activities, that enhance population densities of vector mosquitoes (heavy rains followed by floods, irrigation, higher than usual temperature, or formation of ecologic niches that enable mass breeding of mosquitoes) could increase the incidence of West Nile fever.
Abstract: Devastating floods occurred after heavy rains at many places along the Morava river, Czech Republic, in July 1997. Populations of mosquitoes increased abruptly in the flooded areas thereafter. A total of 11,334 female mosquitoes (9,100 Aedes vexans, 917 Ae. cinereus, 11 Ae. cantans, 1,074 Ae. sticticus, 232 Culex p. pipiens) were collected between July and September 1997 and tested for virus in 117 monospecific pools by intracerebral inoculation of suckling mice. Seven virus isolates were obtained: six were identified as the bunyavirus Tahyna, California group (5 from Ae. vexans, one from Ae. cinereus), and one (strain 97-103 from 57 C. p. pipiens collected at Lanzhot, 48 40'N, 16 56'E, on 17 September) was determined to be the flavivirus West Nile (WNV). Blood samples were obtained from 619 persons being examined for various complaints at hospital and outpatient clinics in the Breclav area between 23 June and 29 September 1997. Sera were assayed by plaque-reduction neutralization (PRN) for antibodies against WNV strains Eg-101 and 97-103. All reacting sera were additionally tested against Central-European encephalitis (CEE) virus by PRN. Antibodies neutralizing WNV were detected in 13 (2.1%) individuals. None of these persons recalled suffering from CEE in the past, or being subjected to vaccination against CEE and yellow fever, and titres of PRN antibodies to CEEV were all below 1:16. Only two of seropositive persons had been abroad during the last 5 years: one in Croatia in 1996, and another lived in South Australia between 1951 and 1994. Paired serum samples had been obtained from 72 persons of the group of 619 people examined. A significant increase (4 times) in antibody titer against WNV between the first (acute-phase) and second (convalescent-phase) samples were detected four times: in two of 41 children and in two of 31 adults. Clinical symptoms compatible with WN fever (WNf) among the four seroconverting persons were only seen in the two children with fever, sore throat, headache, muscle ache, pronounced fatigue, nausea, vomiting, maculopapular rash (including flushed face), slightly enlarged inguinal lymph nodes. The illness lasted for about 7 d., recovery was complete after another week. Of the remaining 9 seropositive persons lacking paired serum samples, one was affected with pronounced headache, muscle ache, prolonged fatigue, nausea, pain on the movement of eyes, maculopapular rash and insomnia in summer of 1997. Two other persons had 'summer fever' with sore throat, lymphadenitis, headache (combined with pain on the movement of eyes in one) in 1997. The other seroreactors did not report any significant illness. The clinical symptoms in 5 persons are compatible with WNf. These are the first reported human cases of WNf in Central Europe (2 confirmed, 3 probable).
Abstract: Faecal samples collected from 308 wild birds of 25 species and 19 rodents of 3 species in South Moravia (Czechland) were pre-incubated in Müller-Kauffmann tetrathionate broth at 42 degrees C for 24 h and then streaked onto Rambach agar plates which were incubated at 37 degrees C for 48 h. Seventeen out of 22 isolates forming orange-red colonies on Rambach agar were identified as Pseudomonas stutzeri, the rest as Pseudomonas sp. and Alcaligenes sp. The colonies of P. stutzeri were either dry, wrinkled and adherent to the agar (resembling Bacillus) or smooth, less adherent (mimicking Salmonella). P. stutzeri was recovered from five species of vertebrates caught in farmland habitats: the house sparrow (Passer domesticus), the tree sparrow (P. montanus), the great warbler (Acrocephalus arundinaceus), the wood mouse (Apodemus sylvaticus) and the common vole (Microtus arvalis). The overall isolation rate was 4.5% in birds (12.6% in house sparrows) and 15.8% in rodents. The procedure can be useful for the isolation of P. stutzeri in clinical and environmental studies.
Abstract: Three strains of Borrelia burgdorferi sensu lato, representing three human pathogenic genomospecies (B31, B. burgdorferi sensu stricto; BR14, B. garinii; BR75, B. afzelii) were grown in BSK-H medium at different temperatures and the spirochaetal cells were counted by dark-field microscopy after 0, 4, 8, 16 and 48 days. Approximate optimum (minimum-maximum) temperatures for the in-vitro growth were found to be 33 degrees C (22-39 degrees C) in strain B31, 35 degrees C (20-40 degrees C) in strain BR75 and 37 degrees C (20-41 degrees C) in strain BR14. Maximum, optimum and minimum growth temperatures seem to be important characteristics of B. burgdorferi s.l. strains, with relevance for the symptomatology, epidemiology and epizoology of Lyme borreliosis.
Abstract: In July 1997, catastrophic floods occurred after long, heavy rains at many places along the River Morava (March) and at its confluence with the River Dyje (Thaya) in Moravia, Czech Republic. We decided to assess the risk of mosquito-borne virus transmission during and after the floods, because the local populations of culicine mosquitoes increased markedly in July and August. Until September 1997, we examined (by i.c. inoculation in ICR newborn mice) 11,334 female mosquitoes in 117 monospecific pools of five species (9100 Aedes vexans in 93 pools, 917 Ae. cinereus in 16 pools, 11 Ae. cantans in 1 pool, 1074 Ae. sticticus in 3 pools, and 232 Culex pipiens in 4 pools) collected in five localities of the district Breclav: Lanzhot, Breclav, Stara Breclav, Tynec a Kostice. Seven suspensions yielded virus: 97-20 (52 Aedes vexans, coll.Breclav, July 31); 97-27 (100 Ae. vexans, coll. Breclav, July 31); 97-28 (45 Ae. cinereus, coll. Breclav, July 31); 97-30 (80 Ae. vexans, coll. Lanzhot, Aug. 19); 97-41 (100 Ae. vexans, coll.Breclav, July 31); 97-46 (100 Ae. vexans, coll. Breclav, July 31); 97-103 (57 Culex pipiens, coll. Lanzhot, Sep. 17). All isolates were passaged twice in suckling mice (SM). The infectious brain homogenates were then used in the constant serum-tenfold diluted virus neutralization tests on XTC cells in flat-bottomed 96-well microplates. Immune mouse sera or ascitic fluids were prepared against the viruses Tahyna, Sindbis and West Nile, inactivated (30 m/56 C) and diluted 1:5. The results were expressed as log neutralization indices (log NI) vs. normal mouse serum. The first six strains were identified as Tahyna virus (log NI was 3.5 to 4.2 against Tahyna T16 immune serum), whereas the last isolate (97-103) was found to be West Nile virus (WN): log NI was 0.2 with Tahyna antiserum, 0.2 with Sindbis antiserum, 3.5 with WN antiserum, and 2.9 with homologous antiserum. The original 97-103 suspension killed 7 of 11 SM on the days 12 and 13 post inoculation, but the average survival time of SM decreased to 8.3 (7 to 9) days in the second passage of the virus. This is the second isolation of WN from mosquitoes in Central Europe; the collection site (48 40'N, 16 56'E) is situated on the flat left bank of the River Dyje close to the Austrian village of Rabensburg (lying on the opposite river bank). WN was previously recovered from Aedes cantans collected at Malacky, W.ţSlovakia, in
June 1972. The ecosystem at Lanzhot (floodplain forest and meadows) is remarkably similar to that at Malacky (including the mosquito and bird fauna), and the air distance between the two localities is only about 25Å£km. WN was previously also isolated from migratory birds of several species in S. and E. Slovakia.
Abstract: Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0-11%), 10.8% (2-43%) and 17.4% (3-58%) for larvae (n = 5699), nymphs (n = 48,804) and adults (n = 41,666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17-20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed.
Abstract: Growth of Borrelia burgdorferi sensu stricto (prototype strain B-31) was studied in Barbour-Stoenner-Kelly BSK-H liquid medium, supplemented with 4.5% rabbit serum and antibiotics (phosphomycin, rifampicin), at various temperatures to early stationary growth phase. The number of cells was determined by darkfield microscopy. In the range of cultivation temperatures of 25 degrees C to 37 degrees C, generation time was between 8.26 and 12.36 h (the shortest one at 33 degrees C), and the specific growth rate between 0.056 and 0.083 h-1 (the highest one at 33 degrees C). The optimum growth temperature for B. burgdorferi was 33 degrees C, although good growth was also observed at 28 degrees C, 30 degrees C, 35 degrees C and 37 degrees C. The strain grew well but slowly at the temperature of 25 degrees C, whereas no growth was observed at 20 degrees C.
Abstract: A total of 1163 I. ricinus ticks were collected in 3 different regions (15 localities) in Styria (Austria) in June 1997 and examined for the presence of spirochetes by dark field microscopy. The mean infection rate was 20.8%. Among 310 adults, 24.2% were positive and among 853 nymphs, 19.6% were positive. All 15 collection areas were shown to harbour infected nymphs with a positivity rate ranging from 5.8% (3/52) to 32.1% (18/56). Isolation attempts in BSKII medium resulted in 29 isolates. Species identification by PCR-RFLP analysis revealed 16 strains of B. garinii, 10 strains of B. afzelii and 2 strains of B. burgdorferi s. s. One isolate showed a mixed population of B. garinii and B. afzelii. In two collection areas, all three major Borrelia species were shown to be present in the tick population.
Abstract: Adiaspores of Emmonsia crescens were found in the lungs of 62.1% of 87 adult rodents from 10 windbreaks compared to only 8.2% of 184 adult rodents caught in 10 adjacent arable fields in South Moravia, Czechland. A significantly higher mean weight proportion of plant remnants (predominantly small roots) was present in the soil from windbreaks (0.74%) than from fields (0.24%). In addition, mean relative abundance of rodents was significantly higher in windbreaks than in fields, and the windbreak soil was more acidic (pH 6.2 vs. 6.9, respectively). On the other hand, there was no significant difference in the water content (17.4% vs. 18.5%, respectively). The difference in the infection rate between the two habitats of the agroecosystem could be caused or affected by land use and farming technology.
Abstract: Host-seeking adult Dermacentor reticulatus ticks were examined for the prevalence of Francisella tularensis in an active natural focus of tularemia along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) and adjacent Lower Austria, in four localities of the flood plain forest-meadow ecosystem during the spring of 1996. The ticks were pooled (10 male or female ticks per group) and inoculated subcutaneously in 4-week-old SPF mice. Dead mice were sectioned, the spleens were homogenized in PBS and passaged in mice, and impression smears from the spleens, liver and heart blood were stained by Giemsa. Twenty-five isolates of F. tularenis were recovered from 1098 pooled D. reticulatus: the minimum infection rate (MIR) is 2.3%. MIRs for 629 female and 469 male D. reticulatus were 2.4% and 2.1%, respectively. The prevalence varied according to locality, but did not significantly differ between the Moravian (2.2%) and Austrian (2.8%) sites. The monitoring of D. reticulatus for F. tularensis might be a valuable contribution to the surveillance of tularemia in Europe.
Abstract: Blood-sucking arthropods, collected in South Moravia, Czech Republic, were examined by darkfield microscopy for borreliae from 1988 to 1996. Among host-seeking ixodid ticks (8481 Ixodes ricinus (L.), 372 Dermacentor reticulatus (Fabr.), 167 Haemaphysalis concinna Koch), borreliae were only observed in adult (23.2%), nymphal (17.2%) and larval (6.3%) I. ricinus. The prevalence of borreliae in I. ricinus did not vary considerably among habitats except for lower values in agroecosystems, xerothermic oak woods and grasslands. The frequency of intensity of spirochaetal infection (log10 counts of borreliae per tick) in I. ricinus approximated the negative binomial distribution. The proportions of host-seeking female and nymphal ticks containing > 100 borreliae were 5.0% and 1.7%, respectively. Among preimaginal ticks (749 I. ricinus, 222 D. reticulatus, 82 H. concinna) parasitizing free-living forest birds and small mammals, borreliae were detected in 6.1% of larval and 10.3% of nymphal I. ricinus, and in one larval H. concinna; 3.2% of the birds and 19.4% of the mammals carried infected ticks. Among 3464 female mosquitoes (Culicidae) of 6 species, 4.1% contained spirochaetes: 1.4% of Aedes vexans Meig., 1.3% of A. cantans (Meig.), 2.2% of A. sticticus (Meig.), 2.2% of Culex pipiens pipiens L. and 5.9% of C. p. molestus Forskal. Borreliae were also detected in 8.4% of 142 fleas (Siphonaptera, largely Ctenophthalmus agyrtes Heller and Hystrichopsylla talpae Curtis) collected from small mammals. Twelve isolates of B. burgdorferi sensu lato have been identified to genospecies: 6 strains from I. ricinus (4 Borrelia garinii Baranton et al., 1 B. afzelii Canica et al. and 1 B. lusitaniae Le Fleche et al.), 1 strain from A. vexans (B. afzelii), 2 strains from C. agyrtes (B. afzelii), and 3 strains from host rodents (B. afzelii).
Abstract: During the years 1993-1995, a total of 3580 culicine mosquitoes of six species were collected in South Moravia, Czech Republic, and examined by dark-field microscopy for the presence of borreliae. Females of Aedes cantans, Ae. sticticus, Ae. vexans, Culex pipiens and Cx pipiens biotype molestus (but not Ae. geniculatus or Culiseta annulata) harboured spirochaetes, the frequencies ranging from 0.7% to 7.8%. One isolate (BR-53) from Ae. vexans was identified as Borrelia afzelii genospecies. The potential role of mosquitoes in the epidemiology of Lyme borreliosis should be investigated.
Abstract: In total, 9,167 mosquitoes (Aedes spp.) and 1,987 ixodid ticks--1,423 Dermacentor reticulatus (F.), 504 Ixodes ricinus (L.), and 60 Haemaphysalis concinna Koch--were examined in an active enzootic focus (floodplain forest-meadow ecosystem) of tularemia in South Moravia. Czech Republic. Although no F. tularensis was detected in mosquitoes or H. concinna, 30 isolates were recovered from D. reticulatus (infection rate, 2.1%) and 1 isolate from I. ricinus (infection rate, 0.2%). Ixodid ticks, especially D. reticulatus, but not mosquitoes serve as vectors (and perhaps reservoirs) of F. tularensis at this natural focus.
Abstract: Cloacal examination of 41 juvenile black-headed gulls (Larus ridibundus) by cultivation demonstrated Campylobacter jejuni in 26 (63%) and Salmonella typhimurium in 21 (51%) of them. All the bird samples were collected in a breeding colony near the town Hodonin, South Moravia, Czech Republic in 1996. Twenty six Campylobacter isolates were tested for antibiotic and drug sensitivity: all were resistant to at least three agents (Penicillin, Tetracyclin and Sulfomethoxazol-trimethoprim) while all were sensitive to Augmentan, Cefotaxim, Ciprofloxacin, Erythromycin, Nitrofurantoin and Cephazidine. Four percent of isolates were resistant to Ampicillin and Nalidixic acid. Of the 21 S. typhimurium isolates tested, 33% were sensitive to all drugs assayed, proportions of the strains resistant to Sulfomethoxazol-trimethoprim, Tetracyclin and Streptomycin were 58%, 16% and 8%, respectively.
Abstract: Cryptococcus neoformans disseminated into the central nervous system (CNS) of intraperitoneally inoculated adult ICR mice, but did not potentiate penetration of concurrently given Bhanja virus (Bunyaviridae) into the CNS. Likewise, Bhanja virus infection did not affect significantly the course of murine cryptococcosis. However, formalin-killed C. neoformans cells non-specifically increased production of antibodies against the virus infection in the animals.
Abstract: A total of 1098 host-seeking adult Dermacentor reticulatus ticks were examined in an active enzootic focus (floodplain meadow and forest ecosystem) of tularemia along the lower reaches of the Thaya (Dyje) river in South Moravia (Czech Republic) and adjacent Austria during the spring of 1996. Twenty-five isolates of Francisella tularensis were recovered (i.e., the overall minimum infection rate was 2.3%). The prevalence rate varied between 0.6% and 3.5% among four localities examined (two in Moravia and two in Austria) but the difference in the rate between Moravia (2.2%) and Austria (2.8%) has not been significant. The results show that D. reticulatus plays the role of an important vector in this extensive, perennial natural focus of tularemia.
Abstract: The survey is based on a total of 1263 records (738 isolations and 525 molecular DNA detections) of five Borrelia burgdorferi s.l. genomic groups available from 26 European countries: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana (= VS116) and B. lusitaniae (= PoTiB2). It shows the geographic distribution, the source (ixodid ticks 802 records, fleas 2 records, mosquitoes 2 records, wild mammals 66 records, human patients 391 records) and the association of the genomic groups with particular clinical manifestations of Lyme borreliosis in humans (B. afzelii significantly prevails in skin lesions whereas B. garinii is more often associated with neuroborreliosis). The most frequent genomic groups in Europe are B. garinii (501 records) and B. afzelii (469 records). They occur across the continent and islands, whereas the third frequent genomic group, B. burgdorferi s.s. (201 records), has only rarely been isolated in eastern Europe. The remaining genomic groups, i.e. B. valaisiana (85 records) and B. lusitaniae (7 records) have only been isolated from, or detected in, Ixodes ricinus ticks in a few European countries.
Abstract: The proposed method consists in examining individual host-seeking vector ticks of the Ixodes ricinus complex by microscopy for borreliae and evaluating the proportion of ticks with more than 100 borreliae. The investigations were carried out in a deciduous oak forest habitat in South Moravia, Czech Republic, over four years (1991-1994). In May (i.e., at the peak of seasonal activity of I. ricinus), about 150 nymphal and 200 adult ticks were examined each year. It was found that annual incidence of human Lyme borreliosis in the region correlated better with the proportion of heavily infected ticks than with the overall infection rate of the ticks.
Abstract: Of 411 forest birds of 33 species examined near Valtice, Czech Republic, 29% were infested with Ixodes ricinus (L.); 2.2% were parasitized by Haemaphysalis concinna Koch. Borreliae were detected in 5.1 and 11.7% of larval and nymphal I. ricinus, respectively. None of the 13 H. concinna tested was infected. In total, 3.2% of the birds examined were parasitized by I. ricinus immatures infected by borreliae. Borreliae-containing ticks parasitized European robin, Erithacus rubecula (L.); Eurasian blackbird, Turdus merula L.; Blackcap, Sylvia atricapilla (L.); Eurasian chiffchaff, Phylloscopus collybita (Vieillot); Great tit, Parus major L.; and Eurasian jay, Garrulus glandarius (L.). The isolate BR-34 from a nymphal I. ricinus off a Eurasian blackbird had a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein pattern, reactions to monoclonal antibodies, and fragments of HindIII digested DNA probed with fla and ospA genes that suggested to us that it belongs to the genospecies Borrelia garinii. Free-living birds may be involved in the circulation of B. burgdorferi sensu lato principally as disseminators of infected ixodid ticks to new area.
Abstract: A total of 924 questing Dermacentor reticulatus (Fabricius), 504 Ixodes ricinus (L.), sixty Haemaphysalis concinna Koch and 718 mosquitoes (Aedes spp.) were examined in a floodplain forest ecosystem during the 1994-95 outbreak of tularaemia in South Moravia, Czech Republic. Francisella tularensis was not isolated from H.concinna ticks or Aedes spp. mosquitoes, whereas twenty-one isolates were recovered from the other haematophagous arthropods. Dermacentor reticulatus revealed a significantly higher infection rate (2.6%) than I.ricinus (0.2%). This tick species acts as principal vector for tularaemia in the enzootic focus. Monitoring of D.reticulatus for F.tularensis thus seems to be a very efficient approach in the surveillance of tularaemia in the flood-plain forest ecosystems of Europe.
Abstract: Twenty isolates of Central European encephalitis (CEE) virus were compared with 20 isolates of louping-ill (LI) virus in indirect immunofluorescence test (IIFT), using a panel of 17 monoclonal antibodies (MoAbs) prepared against the prototype LI virus. Three Asian members of the tick-borne encephalitis (TBE) complex were also included in the comparison: Turkish sheep encephalitis (TSE), Russian spring-summer encephalitis (RSSE) and Langat (LGT) viruses. Antigenic relationships of the viruses were evaluated by Dice similarity coefficient and cluster analysis. The results revealed antigenic heterogeneity of LI isolates, antigenic homogeneity of CEE isolates, and indicated that CEE and LI are related varieties of Eurasian TBE flavivirus that also includes TSE and RSSE strains.
Abstract: The lung tissue of 1143 rodents of five species, caught at a number of sites and habitats in an agro-ecosystem (southern Moravia, Czech Republic) in 1988-1993, was examined for the presence of adiaspores of Emmonsia parva var. crescens (Emmons et Jellison) van Oorschot. The overall prevalence of adiasporomycosis was 16.6%, but its distribution varied significantly according to rodent species (Clethrionomys glareolus 37.6%, Apodemus flavicollis 33.3%, A. sylvaticus 21.1%, A microps 9.2%, Microtus arvalis 2.7%) and habitat (lucerne fields 2.8%, fields with other crops 10.6%, windbreaks 33.1%, woods 15.8%). Wooded areas have consistently higher prevalence rates of rodent adiasporomycosis than arable fields. Some windbreaks could form natural foci of adiasporomycosis in the agricultural, largely deforested landscape.
Abstract: Cloacal swabs, collected from 756 wild synanthropic and exoanthropic birds of 57 species in the Czech Republic, yielded 32 strains of Salmonella typhimurium [phage types (PT) 141, 104 and 41], six isolates of S. enteritidis (PT 8, 4 and 6e), and one each of S. panama and S. anatum. Except for one S. enteritidis isolate from a grey-lag goose (Anser anser) and one S. typhimurium isolate from a coot (Fulica atra), all of the other strains were derived from black-headed gulls (Larus ridibundus), of which 24.7% were found to be infected. The black-headed gull might play a role in the dispersal of pathogenic salmonellae.
Abstract: Ixodes ricinus ticks were collected by flagging vegetation of a mixed oak forest in South Moravia (Czech Republic) at regular two-month intervals from March 1991 to March 1992 and examined for borreliae by darkfield microscopy. Mean annual proportions of infected ticks were 17.2% (15.4% to 21.2% monthly) in females (F), 18.6% (11.8% to 25.9%) in males (M), and 16.3% (12.4% to 20.9%) in nymphs (N); the differences among monthly values were insignificant. However, monthly proportions of intensively infected ticks containing more than 100 borreliae fluctuated widely, from 0.0% to 7.7% (annual mean 3.3%) in F, from 0.0% to 5.6% (mean 2.4%) in M, and from 0.0% to 5.7% (mean 1.9%) in N; the differences among months were significant, and the peak prevalence values were found in July (F, M) or November (N). Mean numbers of borreliae per infected tick reached their peak in September for both adult and nymphal ticks while they were generally low in spring. The highest risk of infection with tick-borne borreliae for vertebrates (including man) occurred in the study area during the month of July; in that month, one either female or nymphal I. ricinus containing more than 100 borreliae was encountered, on the average, every 92 minutes of flagging.
Abstract: The authors describe two cases of tick-born encephalitis family epidemies in the natural focus of tick-born encephalitis (TbE) in the central part of Povazie in the district of Povazká Bystrica in the years 1989 and 1993. The infection took place due to consumption of raw goat milk. The best prevention against tick-born encephalitis is represented by vaccination. (Tab. 4, Ref. 6.)
Abstract: Annotated review on all microorganisms (viruses, bacteria, fungi, protozoa) pathogenic to homoetherm vertebrates, associated with wild birds.
Abstract: Between 1986 and 1991, sera were collected from 33 roe deer (Capreolus capreolus), 24 red deer (Cervus elaphus), four fallow deer (Dama dama), two mouflon (Ovis musimon), 34 wild boars (Sus scrofa), and 48 hares (Lepus europaeus) shot in two areas of the Czech Republic. Collectively, the sera contained antibodies to Coxiella burnetii (prevalence of 12%), Francisella tularensis (4%), Brucella spp. (2%), Central European tick-borne encephalitis virus (8%), Tahyna (California serogroup) virus (36%), and Calovo (= Batai) virus (23%). We propose that these mammals may play a role in maintaining natural foci of Q-fever, Tahyna fever and Calovo virus infection.
Abstract: Cormorants (Phalacrocorax carbo, Pelecaniformes) caught in southern Moravia (Czech Republic) in 1989-1990 were examined for arbovirus infections. Isolation experiments were carried out using blood samples of 56 birds. The results were negative. Serological examinations of 31 birds were performed by haemagglutination-inhibition test (HIT) using 5 arboviral antigens of the genera Alphavirus (Sindbis--SIN) and Flavivirus (tick-borne encephalitis--TBE, West Nile--WN) and of the family Bunyaviridae (Tahyna--TAH, Calovo--CVO). Antibodies were detected only to viruses SIN, WN and TAH at different frequencies: 9.7%, 9.7% and 22.6%. The titres ranged from 1:20 to 1:80.
Abstract: Eperythrozoon was detected in suckling mice inoculated with a suspension of nymphal Ixodes ricinus ticks. The organism was filterable through 220 nm Millipore membranes, moderately sensitive to diethyl ether, pathogenic to suckling but not to adult mice when given intracerebrally, intraperitoneally or subcutaneously, and mimicked an arbovirus. Eperythrozoon should be considered as an agent potentially interfering with experiments performed on laboratory mice.
Abstract: A strain of Eperythrozoon (Rickettsiales: Anaplasmataceae) was isolated from suckling SPF mice (SM) inoculated with a homogenate of nymphal Ixodes ricinus (L.) ticks. The strain, passaged in the SM brain, was partially filterable through a 220-nm Millipore membrane, moderately sensitive to diethylether, and pathogenic for SM while no symptoms were observed in inoculated adult mice or male guinea pigs. A conspicuous cytopathic effect was produced in PS and SPEV (embryo pig kidney) stable cell lines. Both the cell cultures and SM could be used for the infectivity titration of the agent.
Abstract: Stability of type-C botulinum toxin at pH 1.8-12.0 and during exposure to 5 and 28 degrees C for 20 and 16 h, respectively, was tested by titration on adult mice. The toxin was found in the samples kept at pH of 2.7-10.2, whereas, at the pH extremes of 1.8 and 12.0, it was inactivated.
Abstract: A total of 3,254 adults of Ixodes persulcatus tick were collected in a taiga forest habitat situated in the Amgun river basin (Khabarovsk region, the Far East, USSR) and examined individually for the presence and amount of tick-borne encephalitis virus. The over-all proportion of infected ticks was 6.6% and it varied between 3.4% and 9.4% in the years 1982 to 1985. The amount of virus per tick was approximated by the gamma distribution determining a probability that the number of plaque-forming units (PFU) per tick is not greater than a selected value. The frequency distribution of infected ticks followed a model of the negative binomial distribution, enabling the estimation of probability of the occurrence of a given number of infected ticks in the area. However, the parameters of both probability models (i.e., the PFU content per tick, and the frequency of infected ticks) varied for particular years.
Abstract: During a 7- year period were hunted small wild living mammals and examined by ELISA and RIA techniques for the presence of hantavirus antigen and/or antibodies by MFA. In total 3,050 animals of 16 species caught in 9 out of 10 regions of Czechoslovakia, were examined. The proportion of positive animals was 4.4%. To the positive ones with the serotype 2 (Western type) belonged the following: M. arvalis, C. glareolus, P. subterraneus, A. sylvaticus, A. flavicollis. To the Eastern serotype: A. agrarius in eastern Slovakia, A. flavicollis in North of Bohemia and A. sylvaticus in South of Moravia. The repeatedly examined localities were found to be either repeatedly positive or repeatedly negative. The antigen titres in the lungs of M. arvalis were constant irrespective of sex and season of capture. They were, however, much higher in young animals, whereas the proportion of positivities was higher in adults ones. The titres of antigen in the lungs of C. glareolus never exceeded those of M. arvalis.
Abstract: Diverse samples were examined at a site of water-bird mortality, caused by Clostridium botulinum type C toxin in southern Moravia (Czechoslovakia). The toxin was detected in high concentrations in mute swan (Cygnus olor) carcasses (less than or equal to 1 x 10(6) LD50/g) as well as in necrophagous larvae and pupae of the blow flies Lucilia sericata and Calliphora vomitoria (less than or equal to 1 x 10(5) LD50/g) collected from them. It was detected in lower concentrations (less than or equal to 1 x 10(3) LD50/g) in other invertebrates (ptychopterid fly larvae, leeches, sow-bugs) associated with these carcasses, and occasionally in water samples (8 LD50/ml) close to the carrion. The toxin was not detected in the samples of water, mud or invertebrates collected at a distance greater than or equal to 5 m from the carcasses. The toxin-bearing larvae of L. sericata and C. vomitoria, containing 80,000 LD50/g of type C toxin, were exposed in the mud at the study site for 131 days from November to March. Although the toxin activity decreased 25-fold and 40-fold in the two samples of maggots exposed during this period, it remained very high (less than or equal to 3,200 LD50/g). Birds ingesting a relatively low number of these toxic larvae (or pupae) in the spring could receive a lethal dose of the toxin.
Abstract: The prevalence of borreliae in 209 nymphal and 251 adult Ixodes ricinus was investigated in two areas of southern Moravia, Czechoslovakia, using the dark-field and the Giemsa stained-smear techniques. The proportions of infected ticks were 3.8% in nymphs and 10.6% in adults of area A, while they were 29.1% in nymphs and 35.9% in adults of area B. The mean number of borreliae per tick was about 3 to 10 times greater in adult than in nymphal I. ricinus. The results indicate a significant role of nymphal I. ricinus in the ecology of Borrelia burgdorferi and in the epidemiology and epizootiology of Lyme borreliosis.
Abstract: A total of 14,250 haematophagous biting midges (genus Culicoides, Diptera: Ceratopogonidae) were collected in June 1986 in the mountains Ceskomoravská vysocina (about 600 m above sea level); from these 2 strains of Tahyna virus (serogroup California, Bunyaviridae) were isolated. To our knowledge this is the first isolation of a California serogroup virus from the species of family Ceratopogonidae as well as the first report of arbovirus isolation from biting midges in Europe.
Abstract: A total of 378 adult Ixodes ricinus ticks were collected by flagging vegetation in four localities of two districts (Breclav, Znojmo) in south Moravia and examined microscopically. Borreliae were identified in Giemsa-stained midgut smears from 32 (i.e. 8.5%) ticks (9.4% females, 7.2% males); the infection rate varied between 0.0 and 11.4% in the four localities examined. Among female ticks, significantly more were found to be infected in autumn (19.7%) than in spring (5.8%). Dark-field (DF) and Giemsa-stained smears (GS) examinations were compared for their sensitivity in detecting borreliae in 128 field-collected ticks; GS method showed a little higher sensitivity (11.7% ticks were positive) than DF procedure (9.4% ticks positive). Two strains of Borrelia burgdorferi were isolated from a total of 150 adult I. ricinus ticks cultured in BSK medium.
Abstract: A pathogenic agent designated AV 172 was isolated from the blood of a Reed Warbler (Acrocephalus scirpaceus) out of 767 samples from birds belonging to 35 species and 14 families. The birds (largely wetland passerines) were captured in the reed-belt littoral of Nesyt fishpond in southern Moravia, Czechoslovakia, during the years 1984 to 1987. Virus AV 172 has been found to represent probably a new species (designated virus "Sedlec") of family Bunyaviridae. Sedlec virus is pathogenic to suckling and adult mice when inoculated intracerebrally (i.c.) but not intraperitoneally (i.p.) and its ether-sensitive spherical particles measures 90-100 nm.
Abstract: A total of 26,478 ixodid ticks (935 pools) were examined by intracerebral inoculation of suckling mice. Six species of ticks were tested: Ixodes ricinus (23,470 individuals), I. trianguliceps (12), Haemaphysalis punctata (831), H. concinna (39), Dermacentor reticulatus (69) and D. marginatus (2,057). The ticks were collected largely by flagging vegetation, a substantial minority (4%) from animals. Three strains of Francisella tularensis were isolated, one each from I. ricinus (males, district Breclav, southern Moravia), D. reticulatus (males, district Breclav) and D. marginatus (engorged females collected from sheep in Roznava district, eastern Slovakia). D. marginatus and D. reticulatus represent new vector species for Czechoslovakia.
Abstract: Seven strains of the causative agent of Lyme borreliosis have been isolated from ticks of the species Ixodes ricinus (L.) obtained at the territories of Leningrad Province, Lithuania and Czechoslovakia. The strains have been studied in reactions with 5 types of monoclonal antibodies. All isolated strains belong to serotype II according to Barbour's system and are typical for European strains.
Abstract: The authors revealed by means of the haemagglutination-inhibition test (HIT) and the plaque reduction neutralization test (PRNT) that in 1983 to 1985 pregnant women in the Breclav district became infected with the Tahyna virus. HI antibodies against the virus were detected in 96 (28.4%) women from a total of 338 examined. A new infection was proved in 15 (4.4%) women. The majority of infections was contracted in August to October. Positive results were supported by the PRNT. Antibodies against arboviruses Sindbis, Calovo, West Nile, tick-borne encephalitis, Bhandza were not detected in any of the women.
Abstract: Migratory birds (swallow, Hirundo rustica; sand martin, Riparia riparia; house martin, Delichon urbica) caught in southern Moravia (Czechoslovakia) in 1984-87 were examined for arbovirus infections. Isolation experiments were carried out using blood samples of 183 birds (52 swallows, 107 sand martins, and 24 house martins). The results were negative. Serological examinations of 136 birds (36 swallows, 86 sand martins, and 14 house martins) were made by haemagglutination-inhibition test (HIT) using 6 arboviral antigens of the genera Alphavirus (Sindbis--SIN) and Flavivirus (tick-borne encephalitis--TBE, West Nile--WN) and of the family Bunyaviridae (Tahyna--TAH, Calovo, CVO, and Bhanja--BHA). Antibodies against all of the tested viruses were detected at different rates: SIN 2.9%, TBE 1.5%, WN 1.5%, TAH 4.4%, CVO 1.5%, and BHA 2.2%. The titres ranged from 1.20 to 1.80.
Abstract: A sterile suspension containing 950 mouse LD50 per ml of type C botulinum toxin was exposed for various periods to different temperatures. The time required for the 99% (hundred-fold) reduction of toxicity was more than 5 years at -70 degrees C or -20 degrees C, 6 months at +5 degrees C, 3 weeks at +20 degrees C, 2 weeks at +28 degrees C, 2 days at +37 degrees C, 9 h at +42 degrees C, less than 30 min at +56 degrees C, less than 20 min at +60 degrees C, and below 5 min at +80 degrees C. The results suggest that Clostridium botulinum type C toxin, if produced in an ecosystem of the mild climatic zone, might persist there over the winter season and cause the intoxication of vertebrates next early spring in the absence of further microbial toxigenesis.
Abstract: One strain of Bhanja virus was isolated from Dermacentor marginatus male ticks collected from sheep in Slovak Karst, eastern Czechoslovakia (48 degrees 31'N, 20 degrees 28'E). In addition, three strains of Brezová virus (subtype of Tribec orbivirus, Reoviridae) were isolated from Ixodes ricinus ticks. The result represents the northernmost isolation of Bhanja virus and, moreover, its first recovery from D. marginatus in Europe. Cross-neutralization plaque reduction test revealed antigenic similarity of the Czechoslovak strain to the south-European isolates of Bhanja virus, but a more distant relationship to virus strains isolated in Africa and Asia.
Abstract: Antibodies were detected in forest workers to tick-borne encephalitis and Tahyna viruses. In patients hospitalized with fever in summer, several cases of tick-borne encephalitis, Valtice fever (Tahyna virus infection), and infection with Tribec virus were demonstrated by examination of paired sera.
Abstract: Mice, guinea pigs and rabbits were infected with several strains of Bhanja virus (Bunya-viridae) intracerebrally (i.c.), intraperitoneally (i.p.), subcutaneously (s.c.), intravenously, perorally, intranasally (i.n.) or per conjunctivae. Mortality followed the infection by all routes tested (i.c., i.p., s.c.) in suckling mice, by i.c. and i.n. routes in adult mice, by i.c. route in adult guinea pigs whereas did not occur in adult rabbits even after i.c. inoculation. Invasivity indices were estimated for seven virus strains in suckling mice: the log average differences were 2.0 (1.1-2.5) between i.c. and i.p., and 1.6 (1.2-2.3) between i.c. and s.c. mean lethal doses. Average survival times of infected suckling mice were also compared between the strains and inoculation routes, at standardized virus doses. Bhanja virus is markedly neurotropic: in all succumbed animals maximum virus concentrations were detected in the brain, while from other tissues (lung, liver, spleen, skeletal muscles, pancreas, lymph nodes) and blood the virus was recovered irregularly and in lower amounts.
Abstract: A total of 295 birds belonging to 19 species of 7 families of wild Passeriformes were examined by haemagglutination-inhibition test. The birds were caught for an international research program "Balt" at the time of autumn migration (August-September 1984). Their blood sera were examined for antibodies against 6 arbovirus antigens of the genera Alphavirus (Sindbis-SIN) and Flavivirus (tick-borne encephalitis-TBE, West Nile-WN) and family Bunyaviridae (Tahyna-TAH, Calovo-CVO and Bhanja-BHA). Antibodies against all studied viruses were detected at different frequencies: SIN 6.4%, TBE 7.1%, WN 9.7%, TAH 16.3%, CVO 12.1%, and BHA 1.0%.
Abstract: A review on the geographic distribution, vectors and hosts of Bhanja virus (Bunyaviridae) is based on reports about: isolations of the virus; antibody surveys. Bhanja virus has been isolated in 15 countries of Asia, Africa and Europe, and antibodies against it have been detected in 15 additional countries. Vector range includes ticks of the family Ixodidae (subfam. Amblyomminae; not subfam. Ixodinae): 13 species of 6 genera (Haemaphysalis, Dermacentor, Hyalomma, Amblyomma, Rhipicephalus and Boophilus) yielded the virus. Bhanja virus has only rarely been isolated from vertebrates (Atelerix, Xerus, Ovis, Bos; possibly bats), though antibodies have been detected frequently in a wide range of mammals (Ruminantia being the major hosts), in several species of birds (Passeriformes, Galliformes) and even reptiles (Ophisaurus apodus). Natural foci of the Bhanja virus infections are of the boskematic type (sensu Rosický), associated closely with pastures of domestic ruminants infested by ticks in the regions of tropical, subtropical and partly temperate climatic zones.
Abstract: Two squirrels aged 16 weeks and three muskrats aged 24 weeks were subcutaneously infected with a dose of 400 SMicLD50 of the extraneurally passaged "236" strain of Tahyna virus. Viremia was detected in one squirrel (48 and 96 hours post infection) and in two muskrats (24 and 48 hours p.i.). Seroconversion was demonstrated by plaque-reduction neutralization test (PRNT) 21 days p.i. in all animals.
Abstract: The serosurveys conducted in the Silica plateau area of the Slovak karst region revealed the presence of specific neutralizing antibody against tick-borne encephalitis (TBE) virus in 18% of local inhabitants (33 examined, mostly goats and sheep farmers), 54% of goats (26 examined), 18% of sheep (120 examined) and 13% of cattle (60 examined), against LipovnÃk (LIP) virus in 30% of inhabitants, 88% of goats, 55% of sheep and 45% of cattle, and against Bhanja (BHA) virus in 27% of inhabitants, 46% of goats, 29% of sheep and 23% of cattle. The results of hemagglutination-inhibition tests with TBE and BHA antigens were analogous. A detailed analysis of these serologic data points to a recent enhancement of the circulation of LIP and BHA viruses and to a very low TBE virus activity in this natural focus of arboviral infections. The immunological surveys of the 32 former "Roznava disease" patients, conducted 25 years after an extensive epidemic of a TBE virus infection that originated in Roznava in 1951, revealed the presence of neutralizing (and also hemagglutination-inhibiting) antibodies against TBE virus in as many as 78% of cases. Antibodies against LIP and BHA viruses were also detectable in the sera of 16% and 9%, respectively, of these individuals. Populations of the ectoparasites examined for the presence of arbovirus comprised 231 Ixodes ricinus, 806 Dermacentor marginatus and 204 Haemaphysalis punctata ticks and 117 specimens of the louse-flies Melophagus ovinus. Two strains of arbivirus that were antigenically related to LipovnÃk and Tribec viruses belonging to a group of Kemerovo viruses were isolated from male and female I. ricinus ticks collected from cattle.
Abstract: Bhanja virus is acid-labile, relatively thermostable, resistant to trypsin and heparin; a complete inactivation was achieved with chloramine B or formaldehyde, while phenol was ineffective, and UV radiation only partially effective.
Abstract: Randombred (ICR) and inbred (C57B1/6) 4-wk-old SPF male mice were infected extraneurally with Bhanja virus (Bunyaviridae) and subjected to various treatments. Immunosuppression with cyclophosphamide (CPA) affected the course of the infection when a higher dose (10(6) suckling mouse intracerebral LD50) of the virus and 2 or 3 injections of CPA (150 mg/kg each) were given: then a part of the animals died due to viral encephalitis, whereas all the CPA-untreated infected mice survived. A dual peripheral infection with Bhanja and Lipovnik (Reoviridae) viruses did not cause any symptomatic response, and the host's humoral antibody was slightly stimulated. When Bhanja virus was given prior to, or simultaneously with, tick-borne encephalitis virus (Flaviviridae), a moderate decrease of the mortality (due to tick-borne encephalitis) occurred. A mixed peripheral infection of mouse with Bhanja virus and Cryptococcus neoformans, did not result in a fatal virus encephalitis of the host, nor was cryptococcosis affected substantially. However, formalin-killed cells of the fungus ("cryptococcin") administered before the extraneural inoculation of Bhanja virus caused an 8-fold increase of antibodies neutralizing the virus; a mild therapeutic or protective effect of cryptococcin on encephalitis after an intracerebral application of Bhanja virus was also observed.
Abstract: Antigenic relationships between the two known members of the Bhanja serogroup (Bhanja virus, 7 strains; Kismayo virus, two strains) were studied by a plaque-reduction cross-neutralization test in Vero cells, using hyperimmune mouse sera. The results were then subjected to a cluster analysis, which showed (in the form of a dendrogram) a considerable antigenic difference between the two viruses. Within Bhanja virus, the European isolates from Haemaphysalis punctata or H. sulcata ticks were antigenically indistinguishable, while the strains isolated in Africa and Asia from other tick species differed slightly from the European strains and also from each other.
Abstract: Seven juvenile sheep were infected subcutaneously (s.c.) or intracerebrally (i.c.) with Bhanja virus. Distinct symptoms of CNS affection (ataxia, pareses) were observed only in the case of i.c. application of a massive dose (10(-7) to 10(9) SMicLD50) of the Bg 336/336 strain. A short-term and low viremia (one to four days p.i.) was ascertained after i.c. infection with a dose of 10(4.7) to 10(9.2) SMicLD50 of the same strain. On the other hand, the high titres of neutralization antibodies were detected even after inoculation of small doses of the virus (e.g. 50 SMicLD50). Unlike the symptomatic response, the immune response of sheep to Bhanja virus is high.
Abstract: In the years 1981 and 1982 when a mass mortality of wild and domestic water birds was observed on the Starý pond, eleven diseases or just died birds were examined by the neutralization test for botulotoxin and four sludge samples for the presence of Clostridium botulinum. C. Botulinum toxin of C type was detected in four wild ducks (Anas platyrhynchos) from September 1981 and July 1982 and in one gull (Larus ridibundus) and July 1982. The highest titres (of mice i. p. LD50/g) of botulotoxin in ducks were 10(6.3) in the intestinal contents, 10(6.1) in the liver tissue and 10(5.1) in the stomach contents. In one duck we detected, besides the C type, a smaller botulotoxin amount of A type. Therefore we can recommend a complete serotypifying for the cases of botulism in water birds. Out of four sludge samples collected in September 1982 one sample was negative, whereas in three samples the C botulotoxin was demonstrated after propagation and one C. botulinum strain of C type was isolated.
Abstract: Sera of 259 persons, 801 sheep, 54 goats and 60 cows from several regions of Czechoslovakia were examined by tube-neutralization test (TNT) and haemagglutination-inhibition test (HIT) for antibodies against Bhanja virus. The results of both assays correlated well, but TNT was a more sensitive test than HIT. Antibodies neutralizing Bhanja virus were detected in 2.7% of persons, 5.1% of sheep, 18.5% of goats and 16.7% of cattle, most frequently in the district Roznava (Eastern Slovakia), where 38.5% of goats, 26.7% of sheep, 16.7% of cattle and 9.5% humans (predominantly goat or sheep breeders) were positive. A recent circulation of the virus in natural foci of that area is revealed by the detection of antibodies in several calves and lambs, which grazed in one year (1982) only. Roznava district is also unique (among the areas examined) in the presence of the tick Haemaphysalis punctata, principal vector of Bhanja virus in Europe.
Abstract: A nonspecific inhibitor of hemagglutination of Tahyna virus was present in acetone-extracted sera of 3-week old ICR mice but in a much lower level in the 3-day old mice of the same strain. This inhibitor did not neutralize Tahyna virus in vitro and did not protect mice against the lethal challenge by this virus. The inhibitor could not be removed by treatment with: acetone; kaolin, trypsin; heating at 56 degrees C for 30 min or 60 degrees C for 20 min, filtration through Seitz EK pads, a successive treatment with either acetone + trypsin, acetone + filtration, acetone + pH 2.0, or trypsin + heating. However, the inhibitory activity was eliminated by a combined treatment with acetone and heat (56 degrees C/30 min) - or vice versa, or by the isolation of gammaglobulin with the use of rivanol. Fractionated precipitation of serum proteins suggested that the nonspecific inhibitor is a lipoprotein, this being supported by a comparison of the lipoprotein content between samples with the inhibitor present and removed. The course of specific globulin (IgG and IgM) production in mice infected with Tahyna virus was studied in hemagglutination-inhibition test (HIT) using the sera treated with acetone plus heat. This combined treatment of sera might also be suggested for use in routine serological surveys (HIT) of small rodents with Tahyna virus antigen.
Abstract: Nineteen strains of yeasts possessing different characteristics were stored in liquid nitrogen and after 5 years phenotypic characters were evaluated and compared with equivalent strains preserved under paraffin oil. All qualitative characters tested remained stable, and quantitative characters varied only within the range of natural variability.
Abstract: Nineteen strains of taxonomically diverse yeast species tested survived freezing and subsequent five-year storage in liquid nitrogen at - 196 degrees C, using a medium M 2 composed of malt extract, yeast extract, peptone, calf serum and dimethyl sulfoxide. Viability of the yeast cultures after long-term storage ranged from 5 to 97% (average 62%) compared with the viability of the cultures prior to freezing. The use of liquid nitrogen refrigeration for preserving yeast cultures is strongly advocated.
Abstract: Blood sera from 185 Czechoslovak citizens, residents of South-Moravia (Znojmo district: 107, Breclav district: 13) and East-Slovakia (Roznava district: 65) regions, were examined for the presence of neutralizing antibodies to Bhanja arbovirus, using PS and Vero cell lines, constant dose of virus and serial serum dilutions. Specific antibody titres greater than or equal to 1:10 were detected in the sera of 10 persons (i.e. 5.4% of all examined), of which 5 men and 4 women were from district of Roznava (13.8% of all examinees in this district) and 1 woman (forest worker) was from district of Znojmo (0.9% of all examinees in this district), but she could not be excluded to get infected while staying abroad (USSR) for a certain period prior to examination. Striking prevalence of seropositivity among the inhabitants of the Roznava district is apparently linked with two circumstances: this area is infested with Haemaphysalis punctata and Dermacentor marginatus ticks (Bhanja virus vector) and all the seroapositive subjects there in contact with goats and sheep. Detection of the human Bhanja virus-specific antibodies is the first one made on the territory of Czechoslovakia; linked with the previously reported seropositivity of goats and sheep in this area it indicates the presence of this arbovirus in the East-Slovakia region.
Abstract: Several wild birds (4 Fringilla coelebs, 1 Coccothraustes coccothraustes and 2 Erithacus rubecula) were inoculated subcutaneously with Bhanja virus (BHAV). No clinical symptoms of infection were observed in any of the birds; a low viremia was demonstrated only in C. coccothraustes (2 and 4 days p.i.), seroconversion in all birds. BHAV was not isolated from organs 32 days p.i. Consequently, the tested birds do not seem to serve as "amplifying hosts" of BHAV. The paper includes a survey of geographic distribution of Bhanja virus and a list of its vectors. A hypothesis on the indirect dissemination of BHAV by birds by means of infected ticks is discussed. According to this hypothesis the European birds could be divided into 3 categories: 1. birds of the Palaearctic-African migration system in which African vectors of BHAV were detected and which could introduce into southern parts of the Palaearctic Region infected ticks from tropical savannahs of Africa (i.e., the biome of main BHAV distribution), 2. migratory birds, hosts of BHAV vectors occurring in southern Europe, which could transfer viruliferous ticks from South to Central Europe, 3. sedentary or migratory birds of steppe or forest--steppe biotopes, hosts of BHAV vectors, which could increase the virus circulation in natural foci either by dispersion of infected ticks to short distances or by a support of their life cycles.
Abstract: Forty-three association (similarity) coefficients were collected and evaluated in this survey. Some of them are synonyms or direct correlates with earlier described indices, others are mere transform from one range of values to another. Several coefficients are incompatible with suggested admissibility conditions of the minimum-maximum value, symmetry, discrimination between positive and negative association, or monotonicity. As a result, 23 coefficients were excluded and the remaining 20 admissible measures were subjected to an empirical trial on interspecific association data among fungi of the genus Chaetomium, with the use of cluster analysis. The classification produced five main clusters of related coefficients. A set of measures that generally work well comprises the coefficients of Jaccard, Dice, Kulczynski, and Ochiai. For some specific purposes, however, other admissible coefficients would be more optimal.
Notes: Reprints available on request from the author.
Abstract: Two young female rabbits (Oryctolagus cuniculus var. edulis) were inoculated subcutaneously with Bhanja virus (BHA) in a dose of 85 intracerebral LD50 for suckling mice (SMicLD50) and 8500 SMicLD50 (rabbit A and B, resp.). In rabbit A no clinical symptoms, nor viremia were observed, only seroconversion was revealed on day 7 p.i. On the other hand, in the rabbit B hypothermy on days 7--11 p.i., a mild adynamia on day 11 p.i. and a slight paresis on one hind leg on days 23--30 p.i. were observed; traces of BHA virus in the blood were detected on day 9 p.i. and seroconversion on day 7 p.i. The virus was not isolated from the interior organs (brain, liver, spleen, kidneys, lungs and heart) of the two rabbits dissected 38 days p.i.
Abstract: Juvenile SPF randombred ICR mice were given intraperitoneally (i.p.) a single dose of cyclophosphamide (CPA - 80 mg/kg), and 10(3.7) SMicLD 50 of Tahyna virus (California group, Bunyaviridae) one day later. The mice survived, but low concentrations of the virus were recovered from their brains 21 days after infection. No virus was detected in the brains of the animals infected but CPA-untreated. A lower body weight gain and a twofold decrease of virus neutralizing (VN) antibody titre were also observed in the CPA-treated mice. When mice were infected i.p. with 10(4.5) SMicLD50 of the virus, and CPA given 2 days (130 mg/kg) and 5 days (160 mg/Kg) later, the animals died of an acute encephalitis (with a mean virus titre at death of 10(7.3) SMicLD50/g brain), while all the infected but saline-treated animals survived, with no virus recovered from their brains. VN antibody was absent in the CPA-treated infected mice compared to high titres of antibodies detected in the CPA-untreated, infected animals.
Abstract: Apparently healthy hair of insectivores and rodents originating from two regions situated at different altitudes in Austria was examined on the presence of dermatophytes. Dermatophytes Microsporum persicolor, Trichophyton georgiae, T. mentagrophytes var. erinacei, T. mentagrophytes var. mentagrophytes and T. terrestre were recovered from the hair of 27 animals (32.9%), belonging to 10 species. The results revealed evident specificity of dermatophytes to host mammals irrespective of the effect of different altitude of localities. The existence of dermatophytes in the healthy hair of mammals is due either to mere contamination from environment (findings of geophilic species) or to inapparent infection (findings of zoophilic species).
Abstract: A total of 488 wild small mammals (16 species) trapped in two federate units of Austria, Steiermark and Burgenland, were examined on the presence of blood parasites. In Steiermark Grahamelles were detected in Neomys fodiens, Clethrionomys glareolus, Microtus arvalis, M. agrestis, Apodemus flavicollis and A. sylvaticus, while Trypanosoma evotomys and Hepatozoon erhardovae were found in C. glareolus, and Babesia microti in Pitymys subterraneus and M. agrestis. In Burgenland Grahamelles were demonstrated in Sorex araneus, C. glareolus, A. sylvaticus, A. flavicollis and Rattus norvegicus, while trypanosoma grosi was encountered in A. flavicollis and Babesia microti in C. glareolus, M. arvalis and A. flavicollis.
Abstract: A total of 308 fungi was isolated from interior organs (lungs, spleen, liver) of 529 small mammals belonging to 21 species, 7 families and 3 orders (Insectivora, Chiroptera, Rodentia), some of these being potentially pathogenic to vertebrates (e.g. Aspergillus flavus, A. fumigatus, Geotrichum candidum, Mucor pusillus, Rhizopus arrhizus). In one vole (Microtus arvalis) captured in South Moravia, adiaspiromycosis (Emmonsia crescens) was demonstrated. Comparison of mycoflora of hair and that of interior organs of wild small mammals revealed that out of the total number of isolates the following fungi were represented in a higher proportion from visceral organs than from the hair: Aspergillus (A. amstelodami, A. flavus, A. repens), Aureobasidium (A. pullulans), Candida, Cladosporium (C. herbarum), Cryptococcus, Fusarium, Gliocladium (G. deliquescens), Helminthosporium, Kloeckera, Mucor (M. fragilis, M. hiemalis, M. pusillus), Paecilomyces marquandii, Penicillium (P. purpurogenum), Phoma, Rhizopus arrhizus, Scopulariopsis (S. candida, S. koningii) and Torulopsis.
Abstract: Six weeks old random bred ICR mice were inoculated intraperitoneally with two different doses of Tahyna virus (125 and 12 500 smicLD50). The higher dose caused marked involvement of the central nervous system in a few animals and induced high virus neutralization (VN) antibody titres in more animals than the lower dose. The dose of inoculum had no effect on the height of VN antibody titre in mice with antibody titres > 64. The proportion of mice with VN antibody titres > 64 was 72.7% in males and 56.8% in females. Male mice thus appeared to be somewhat more susceptible to Tahyna virus infection than female mice.
Abstract: Two plaque variants of Chikungunya (CHIK) virus were serologically compared with O'nyong nyong (ONN) virus in order to elucidate the reported one way antigenic relationships between the two viruses. Three different hypotheses are examined and evidence is shown to support one of them. Comparison of some biological properties of the viruses showed ONN to be distinct in some respects. All viruses and variants were found to replicate in Anopheles gambiae cells.
Abstract: Antigenic relationships among seven California group strains were studied by a plaque-reduction neutralization test (PRNT). Cross-reactions occurred in most cases but three subgroups were noted: (1) the major serogroup contained the viruses of California encephalitis, LaCrosse, Snowshoe Hare and Trahyna (including the Lumbo strain) whereas (2) Jamestown Canyon and (3) Trivittatus viruses were distinct. There was no significant difference between the PRNT results in mammalian (PS) cells incubated at 37 degrees C and amphibian (XTC-2) cells incubated at 28 degrees C. Trivittatus virus failed to produce plaques in XTC-2 cells.
Abstract: Three Putorius eversmanni pole-cats and two Martes foina martens aged about 9 months were subcutaneously infected with about 260 suckling mouse LD50 of the extraneurally passaged "236" strain of Tahyna virus (California group, genus Bunyavirus). Viraemia with maximal titres of 1.32 (pole-cats) and 1.28 (martens) dex intraperitoneal (i.p.) mouse LD50/0.02 ml was demonstrated from 48 to 96 hr after inoculation (p.i.). By the plaque-reduction neutralization test, seroconversion was demonstrated 15 days p.i. (from less than 4 to titres of 8192 in pole-cats and 4096 in martens).
Abstract: Bhanja virus was isolated from ticks of the genus Haemaphysalis (H. punctata and H. sulcata) collected from sheep pastured in the surroundings of Akhtopol in southeast Bulgaria, and simultaneously blood sera of 58 sheep were investigated in plaque-reduction neutralization test with B lranja arbovirus. All sera contained antibodies to this virus in titres from 1:32 to 1:4096 (geometrical mean titre 1:494). The district of Akhtopol proved to be a natural focus of Bhanja virus.
Abstract: 254 pools of 4,115 mosquito larvae belonging to nine species were examined by isolation experiments. The larvae were collected in breeding places in an inundated forest--a natural focus of Tahyna virus, in April, June and July 1974 and 1975. Tahyna virus was isolated from one pool of 10 Culiseta annulata larvae collected in July 1974. Ecological questions concerning this finding are discussed.
Abstract: Inoculum with 125I added as a marker was used to determine the volume actually administered to suckling mice on intracerebral (i.c.) inoculation. In 1-2 days old animals, this volume corresponded to approximately 75% of a 20 microliter dose.
Abstract: All 20 yeast strains of 17 species tested survived 75 days (the length of the experimental period) in liquid nitrogen at -196 C. The components of the more protective of the two freezing media used were (w/v) malt extract 2.5%, yeast extract 0.25%, peptone 0.5%, calf serum 15% (v/v) and dimethyl sulfoxide 10% (v/v). Viability of the cells in this medium after rapid uncontrolled freezing and thawing in selaed plastic ampoules ranged from 2% to 98% (average 67%) compared with the viability of the cultures before freezing. In only 4 strains was survival lower than 50%. (90 references).
Abstract: Three foxes about 18 weeks old were each subcutaneously inoculated with approximately 200 LD50 dose of extraneurally passaged strain of the Tahyna virus "236". Viremia was demonstrated in all animals during the 48-96 hours interval after infection. The maximum value was 1.31 dex LD50/0.02 ml. Three weeks after infection a seroconversion was revealed by means of hemagglutination-inhibition test (from titre less than 1 : 10 to 1 : 320) and plaque-reduction neutralization test (from titre less than 1 : 4 to titre 1 : 4096 or 1 : 8192).
Abstract: SPF white mice were inoculated intraperitoneally with 5 strains of saprophytic fungi of the mycelial genera Chrysosporium (C. keratinophilum, C. tropicum) and Paecilomyces (P. lilacinus, P. marquandii, P. victoriae). The fungi caused granulomatous lesions in the peritoneal cavity and they were recultured (except P. lilacinus and P. marquandii) two months after inoculation. Spores, short hyphae and budding cells of all the fungi were observed in the granulomas stained by periodic-acid Schiff (PAS) and methenamine-silver nitrate (Grocott) techniques.
Abstract: Twelve species of keratin decomposers isolated from 923 samples of feathers, nests and pellets of 90 species of free-living birds were clustered in 4 "econs" (numerical ecological groups): (A) Artroderma tuberculatum, A. ciferrii, A. multifidum, A. cuniculi (associated with the birds nesting in hollows); (B) Ctenomyces serratus, Arthroderma quadrifidum, Chrysosporium evolceanui (moderately alkalophilic species associated with the birds having a frequent contact with the soil); (C) Chrysosporium keratinophilum, Aphanoascus fulvescens, Chrysosporium tropicum (alkalophilic and hygrophilic species); (D) Arthroderma curreyi, Aphanoascus terreus (the species common in water habitats and associated frequently with exoanthropic birds). Two simple numerical procedures were used and compared: (1) Jaccard's association coefficient, and Sörensen's complete linkage clustering; (2) Forbes's association coeficient adjusted with chi-square, and the 3-linkage clustering technique. The method (2) seemed to be more effective.
Abstract: Samples of pigeon droppings were taken from 7 sites in a church tower contaminated with C. neoformans, and the distribution patterns of the fungus were studied. From various sites, 0 to 3 x 10(5) viable C. neoformans particles were recovered per one gram of dry excreta. The factors causing the different density of C. neoformans population in the habitat were: uric acid share of the total nitrogen, creatinine content, sunlight and probably pH. Chemical composition of the substrate is the primary factor in the distribution of C. neoformans in droppings.
Abstract: UV irradiation of pigeon droppings resulted in an increased concentration of some inhibitors (peroxides) of growth of Cryptococcus neofarmans. This may be, in addition to the direct germicidal action of sunshine, another cause of the rare occurrence of this fungus in pigeon droppings on unsheltered sites in natural habitats.
