Igor Majláth

Borrelia lusitaniae and green lizards (Lacerta viridis), Karst Region, Slovakia.

TOC summary line: The green lizard is implicated in the transmission cycle of B. lusitaniae.

Detection of Borrelia burgdorferi sensu lato in lizards and their ticks from Hungary.

To investigate the involvement of lizard species in the natural cycle of Borrelia burgdorferi sensu lato (s.l.) in Hungary, a total of 186 reptiles belonging to three species--126 green lizards (Lacerta viridis), 40 Balkan wall lizards (Podarcis taurica), and 20 sand lizards (Lacerta agilis)--were captured in 2007 and 2008. All ticks removed from the lizards were Ixodes ricinus, either larvae (324/472; 68.6%) or nymphs (148/472; 31.4%). More than half (66/126; 52.4%) of L. viridis individuals were infested, and the prevalence of tick infestation on both the other two species was 35% each. All 472 I. ricinus ticks and tissue samples collected from 134 collar scales and 62 toe clips of lizards were further analyzed for the presence of B. burgdorferi s.l. with polymerase chain reaction. The amplification of B. burgdorferi s.l. DNA was successful in 8% (n = 92) of L. viridis, 9% (n = 32) of P. taurica, and 10% (n = 10) of L. agilis tissue samples. Restriction fragment length polymorphism genotyping identified the species Borrelia lusitaniae in all tested lizard samples. Prevalence of B. burgdorferi s.l. in ticks collected from L. viridis, P. taurica, and L. agilis was 8%, 2%, and 0%, respectively. Most of the infected ticks carried B. lusitaniae (74% of genotyped positives); however, Borrelia afzelii (5%) and B. burgdorferi sensu stricto (21%) were detected in ticks removed from green lizards and Balkan wall lizards, respectively. We conclude that lizards, particularly L. viridis, can be important hosts for I. ricinus larvae and nymphs; thus, they can be regarded as reservoirs of these important pathogen vectors. The role of green lizards has been confirmed, and the implication of Balkan wall lizards is suggested in the natural cycle of B. lusitaniae at our study site.

Anaplasmataceae and Borrelia burgdorferi sensu lato in the sand lizard Lacerta agilis and co-infection of these bacteria in hosted Ixodes ricinus ticks

BackgroundAnaplasmataceae and Borrelia burgdorferi s.l. are important tick-borne bacteria maintained in nature by transmission between ticks and vertebrate hosts. However, the potential role of lizards as hosts has not been sufficiently studied.ResultsThe current study showed that 23 of 171 examined sand lizards Lacerta agilis were PCR positive for Anaplasmataceae. The nucleotide sequences of the several selected PCR products showed 100% homology with Anaplasma spp. found in Ixodes ricinus collected in Tunisia and Morocco (AY672415 - AY672420). 1.2% of lizard collar scale samples were PCR positive for B. lusitaniae. In addition, 12 of 290 examined I. ricinus were PCR positive for B. burgdorferi s.l. and 82 were PCR positive for Anaplasmatacea. The number of ticks per lizard and the number of ticks PCR positive for both microorganisms per lizard were strongly correlated. Moreover, we found a significant correlation between numbers of ticks infected with Anaplasmataceae and with B. burgdorferi s.l. living on the same lizard. However, there was no significant correlation between detection of both bacteria in the same tick.ConclusionsTo the best of our knowledge, this is the first report of Anaplasmataceae DNA and additionally the second report of B. burgdorferi s.l DNA detection in the sand lizard.

Morphological and molecular characterization of Karyolysus – a neglected but common parasite infecting some European lizards

BackgroundBlood parasites of the genus Karyolysus Labbé, 1894 (Apicomplexa: Adeleida: Karyolysidae) represent the protozoan haemogregarines found in various genera of lizards, including Lacerta, Podarcis, Darevskia (Lacertidae) and Mabouia (Scincidae). The vectors of parasites are gamasid mites from the genus Ophionyssus.MethodsA total of 557 individuals of lacertid lizards were captured in four different localities in Europe (Hungary, Poland, Romania and Slovakia) and blood was collected. Samples were examined using both microscopic and molecular methods, and phylogenetic relationships of all isolates of Karyolysus sp. were assessed for the first time. Karyolysus sp. 18S rRNA isolates were evaluated using Bayesian and Maximum Likelihood analyses.ResultsA total of 520 blood smears were examined microscopically and unicellular protozoan parasites were found in 116 samples (22.3% prevalence). The presence of two Karyolysus species, K. latus and K. lacazei was identified. In total, of 210 samples tested by polymerase chain reaction (PCR), the presence of parasites was observed in 64 individuals (prevalence 30.5%). Results of phylogenetic analyses revealed the existence of four haplotypes, all part of the same lineage, with other parasites identified as belonging to the genus Hepatozoon.ConclusionsClassification of these parasites using current taxonomy is complex - they were identified in both mites and ticks that typically are considered to host Karyolysus and Hepatozoon respectively. Furthermore although distortions to the intermediate host erythrocyte nuclei were observed, the defining characteristic of Karyolysus, the haplotypes were nearly identical to those reported from lizards in the Iberian Peninsula, where such distortions were not reported and which were thus identified as Hepatozoon. Based on the phylogenetic analyses, neither vertebrate host, nor geographical patterns of the studied blood parasites could be established.

Polymerase chain reaction confirmation of Babesia canis canis and Anaplasma phagocytophilum in dogs suspected of babesiosis in Slovakia.

Canine babesiosis was considered an imported tick transmitted disease until the first case of autochthonous canine babesiosis in Slovakia was described in 2002. Since then, the number of cases kept increasing every year. The causative agent of babesiosis in dogs is not yet characterized; therefore, the aim of our study was to determine the agent and the rate of infection in the vector tick D. reticulatus in Slovakia. Babesia canis canis was detected in 80 out of 87 blood samples from dogs with clinical manifestations of babesiosis. Six dogs suspected of babesiosis tested positive for presence of Anaplasma phagocytophilum, and one mixed infection of B. c. canis and A. phagocytophilum was detected. B. c. canis was detected in 35.6% questing adults of D. reticulatus. The obtained sequences from blood samples showed 99.7% and from D. reticulatus, 99.4% similarity with the B. c. canis (AY072926) from dogs infected in Croatia. In our study, we characterized the agent of canine babesiosis from blood samples of naturally infected dogs and D. reticulatus, the vector tick. Further, the presence of A. phagocytophilum, bacterium responsible for the canine granulocytic anaplasmosis, was recorded in dogs for the first time in Slovakia.

Blood parasites in two co-existing species of lizards (Zootoca vivipara and Lacerta agilis)

We investigated the occurrence of blood parasites of two lizard species: the common or viviparous lizard (Zootoca vivipara) and the sand lizard (Lacerta agilis) in western Poland. Selected traits of lizard body morphology were studied with respect to the presence and intensity of haematozoan infection in blood samples collected from 218 adult lizards; 88 of the common lizard and 130 of the sand lizard. Haemogregarinid blood parasites were found to be the common parasite of both lizard species in studied locality with prevalence 39.8 (95% CL, 29.5–50.8) for Z. vivipara and 22.3 (95% CL, 15.5–30.4) for L. agilis. Incidence of parasitemia did not differ between sexes and was not correlated with morphological traits or presence of ectoparasites—Ixodes ricinus ticks. However, a significant difference between the two species of lizards was a greater frequency of haemogregarinid parasitism in Z. vivipara.

Phylogenetic relationship of Hepatozoon blood parasites found in snakes from Africa, America and Asia.

The blood parasites from the genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleida: Hepatozoidae) represent the most common intracellular protozoan parasites found in snakes. In the present study, we examined 209 individuals of snakes, from different zoogeographical regions (Africa, America, Asia and Europe), for the occurrence of blood parasites using both molecular and microscopic examination methods, and assess phylogenetic relationships of all Hepatozoon parasites from snakes for the first time. In total, 178 blood smears obtained from 209 individuals, representing 40 species, were examined, from which Hepatozoon unicellular parasites were found in 26 samples (14·6% prevalence). Out of 180 samples tested by molecular method polymerase chain reaction (PCR), the presence of parasites was observed in 21 individuals (prevalence 11·6%): 14 snakes from Africa belonging to six genera (Dendroaspis, Dispholidus, Mehelya, Naja, Philothamnus and Python), five snakes from Asia from the genus Morelia and two snakes from America, from two genera (Coluber and Corallus). The intensity of infection varied from one to 1433 infected cells per 10 000 erythrocytes. Results of phylogenetic analyses (Bayesian and Maximum Likelihood) revealed the existence of five haplotypes divided into four main lineages. The present data also indicate neither geographical pattern of studied Hepatozoon sp., nor congruency in the host association.

PCR detection of re-emerging tick-borne pathogen, Anaplasma phagocytophilum, in deer ked (Lipoptena cervi) a blood-sucking ectoparasite of cervids

Anaplasma phagocytophilum is an obligate intracellular bacterium, circulating in the natural foci in enzootic, vector-host cycle. In Europe, A. phagocytophilum is transmitted by Ixodes ricinus ticks. In Slovakia, cervids which are considered as naturally infected reservoirs of A. phagocytophilum are besides the ticks commonly infested with insects from the family Hippoboscidae. In this study, the presence of A. phagocytophilum was confirmed in deer keds (Lipoptena cervi) removed from deer by using of molecular approach. Detection of A. phagocytophilum in deer keds represents the remains of infected blood meal taken from infected deer host, what underlines the potential role of these blood-sucking insects in the mechanical transmission of pathogenic bacteria within the susceptible population of wild animals. Moreover, it may suggest the risk for the transmission of A. phagocytophilum or related pathogens to humans and healthy animals via the bite of infected hematophagous ectoparasites.

Escape behavior of the green lizard (Lacerta viridis) in the Slovak Karst

The escape behavior of the green lizard (Lacerta viridis) was investigated in natural conditions of the Slovak Karst, Slovakia. The seasonal changes of approach distances and types of escape behavior were monitored for both sexes, also in relation to differences in anthropogenic influence. In our study, the escape behavior of green lizard changed significantly during the season, and differ significantly between localities and in respect to human disturbance. At the beginning of the season, females stayed in close proximity to a refuge and hid immediately after being disturbed. Among males, the distance to and from a refuge was longer and any accessible protrusions were used to hide. The approach distance was greater among males. During the mating period, approach distance for females and males was similar and both sexes hid in a refuge. However, after the mating period, the approach distance for females was decreasing, whereas that of males begun to increase.

Identification of Hepatozoon erhardovae Krampitz, 1964 from bank voles (Myodes glareolus) and fleas in Southern Hungary.

In order to investigate the prevalence and life cycle of apicomplexan parasites, small mammals were live-trapped with modified Sherman traps in Southern Hungary between 2010 and 2012. Altogether, 528 rodents (Apodemus flavicollis Melchior, 1834, Apodemus agrarius Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758 and Micromys minutus Pallas, 1771) were collected and four shrews (Sorex spp.) were by-catched. Captured animals belonging to non-protected species were euthanized, and spleen samples were preserved for histological and molecular analyses. During the examination of spleen smears, Hepatozoon parasites were observed in eight out of 48 bank voles (M. glareolus). DNA was isolated from altogether 221 spleen samples, and 18S rDNA was amplified using two different PCR protocols. The eight bank vole samples were positive with PCR, but none of the other M. glareolus spleen samples or any of the tissue samples from other species were found to be infected. Sequenced amplicons were very similar to Hepatozoon spp. detected in M. glareolus in Spain and Poland. Ectoparasites were collected from the small mammal carcasses and from the vegetation. Hepatozoon DNA was not found in the 181 ticks removed from the small mammals or in the 162 ticks collected with flagging, but was detected in all three flea species (4/43 Megabothris turbidus Rothschild, 1909, 3/10 Ctenophthalmus assimilis Taschenberg, 1880 and 7/78 Ctenophthalmus agyrtes Heller, 1896). Based on gamont morphology, vertebrate and arthropod host species and DNA sequences, the parasites in our study can be identified as Hepatozoon erhardovae.