Determination of <Emphasis Type="Italic">Rhipicephalus</Emphasis> spp. as vectors for <Emphasis Type="Italic">Babesia ovis</Emphasis> in Iran

The tick-borne diseases of livestock constitute a complex of several diseases with different etiological agents, such as protozoa, rickettsia, bacteria, and viruses. One problem discussed in protozoan infection is the determination and characterization of the transmitter agent. Because many analyses were performed with the salivary gland smears using the methyl-green-pyronin staining method or the Feulgen staining method, the transfer vector remains unanswered in some cases. The aim of this study is to recognize Babesia ovis in the salivary gland of Rhipicephalus spp. using polymerase chain reaction (PCR). Deoxyribonucleic acid (DNA) was isolated from 269 salivary gland of Rhipicephalus spp. (108 R. bursa, 87 R. turanicus, 74 R. sanguineus) collected from sheep with suspected to babesiosis. The isolated DNA was then analyzed with the primers derived from the hypervariable region V4 of 18S ribosomal ribonucleic acid (rRNA) of the Babesia species. For the specificity of the PCR product and discriminating from Babesia motasi and Babesia crassa, nested PCR and restriction fragment length polymorphism was performed. As positive control for the DNA extraction procedure, the DNA was analyzed with the common primers designed from the 18S rRNA of the Ticks (Rhipicephalus, Hyalomma, Haemophysalis, Dermacentor, Ixodes, Boophilus). B. ovis was detected in salivary gland of 18.5% R. bursa, 9.1% R .turanicus, and 8.1% R. sanguineus, respectively.     

Determination of prevalence and risk factors for infection with <Emphasis Type="Italic">Babesia ovis</Emphasis> in small ruminants from Turkey by polymerase chain reaction

In this study, PCR and thin blood smear-based diagnostic methods were used to assess the frequency of Babesia infection in small ruminants. A total of 300 sheep and 100 goats from 37 randomly selected herds located in eight locations of eastern Turkey were examined for the presence of Babesia infection and any tick species on the body of the animals. Of 400 blood samples examined, 6 (1.5%) were positive for Babesia spp. piroplasms upon microscopic examination, whereas 33 (8.25%) were positive for the presence of B. ovis by PCR. The prevalence of babesiosis in small ruminants detected by PCR was significantly higher than obtained in microscopic examination of thin blood smears (P < 0.05). Thirty-three animals produced the DNA fragment specific for Babesia ovis of which 32 (10.66%) were sheep and 1 (1%) was goat. The difference between the prevalence of Babesia infection in sheep and goats were statistically significant (P < 0.05). The prevalence of Babesia infection in different age groups of sheep were statistically non-significant (P > 0.05). The frequency of B. ovis infection was higher in herds with tick burden than no tick burden (P < 0.05). Seven amplicons (six from sheep and one from goat) were sequenced. The resulting sequences were identical to the recently reported nucleotide sequence of B. ovis. A total of 510 ticks belonging to the Rhipicephalus spp. were collected from sheep. Ticks were identified to be R. bursa and R. turanicus on the basis of morphological features. Nucleotide sequences data reported in this paper are available in EMBL, GenBank and DDJB databases under the accession numbers: DQ409332, DQ409333, DQ409334, DQ409335, DQ409336, DQ409337, DQ409338.     

Molecular detection of Ehrlichia canis,Anaplasma bovis,Anaplasma platys,Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.     

Molecular detection and identification of Ehrlichia and Anaplasma species in ixodid ticks

A polymerase chain reaction (PCR) assay followed by partial sequencing of the 16S ribosomal RNA gene was performed for the presence of Ehrlichia and/or Anaplasma. A total of 242 ixodid ticks were collected from domestic ruminants and their shelters, as well as humans, and their individual salivary glands were dissected out for DNA. From the 242 ticks analyzed, six (2.47%), comprising three Hyalomma anatolicum anatolicum, one Rhipicephalus bursa, and two Rhipicephalus sanguineus, were positive. Of these sequenced samples directly obtained from the PCR products, three sequences from H. a. anatolicum were identical to that of the gene of Ehrlichia spp. strains. One sequence identified in R. bursa was closely related to Anaplasma platys. The remaining two sequences detected in R. sanguineus were similar to that of the gene of Anaplasma ovis. The study presented here provides preliminary data regarding the presence of rickettsial pathogens in ticks in Turkey. Nucleotide sequence data reported in this paper are available in GenBank, EMBL, and DDBJ databases under accession numbers EU191227–EU191231 and EU191233.     

Molecular detection of Rickettsia massiliae,Rickettsia sibirica mongolitimonae and Rickettsia conorii israelensis in ticks from Israel

Rickettsioses are recognized as important emerging vector-borne infections of humans worldwide. Previous reports documented the presence of two spotted fever group rickettsiae in Israel, Rickettsia conorii israelensis and Rickettsia felis. The aim of this study was to characterize the diversity of rickettsiae in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 131 tick pools, 83 of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (each with 2–10 ticks per pool), were included in this study. In addition, 13 Hyalomma sp. ticks were collected. The ticks were molecularly screened for rickettsiae, targeting the citrate synthase (gltA) and the outer membrane protein A (ompA) gene loci. Rickettsia massiliae ompA DNA (100% sequence identity; 180 bp) was detected in 32 Rh. turanicus and 12 Rh. sanguineus tick pools. R. conorii israelensis was detected in three Rh. sanguineus pools. Rickettsia sibirica mongolitimonae ompA DNA (100% sequence identity; 182 bp) was found in one Hyalomma tick. This study reports the first detection of R. massiliae and R. sibirica mongolitimonae in ticks from Israel. This is the first report describing the presence of these human pathogens in the Middle East.     

Species diversity and geographic distribution of hard ticks (Acari: Ixodoidea: Ixodidae) infesting domestic ruminants, in Qazvin Province, Iran

This report presents the results of the first faunistic study of hard ticks in Qazvin province of Iran. The primary objective was to determine the species diversity and geographic distribution of hard ticks that parasitize domestic ruminants. Information about the abiotic preferences of these species has been provided. A total of 286 cattle, 1,053 goats, and 2,050 sheep were examined in 13 villages in 28 flocks distributed throughout the studied areas. Total direct body collections of ticks were made from each domestic ruminant. A total of 228 Ixodid specimens belonging to nine species in three different genera were recorded in the areas, including Boophilus annulatus (Say, 1821), Hyalomma anatolicum Koch, 1844, Hyalomma asiaticum (Schulze and Schlettke, 1929), Hyalomma detritum Schulze, 1919, Hyalomma dromedarii Koch, 1844, Hyalomma marginatum Koch 1844, Hyalomma schulzei Olenev, 1931, Rhipicephalus bursa Canestrini and Fanz, 1878 and Rhipicephalus sanguineus (Latreille, 1806). The most abundant species on sheep was R. sanguineus (46.92%), while B. annulatus (6.6%) found only on cattle. A finding of great significance was that R. sanguineus, the main vector of babesiosis, is firmly established throughout the counties. A further objective of the study was to compare the abundance of the major tick species on domestic ruminants. This was carried out at 19 sampling sites. The highest number of ticks was collected in July–August during the hot season.     

Molecular and parasitological survey of <Emphasis Type="Italic">Hepatozoon canis</Emphasis> (Apicomplexa: Hepatozoidae) in dogs from rural area of Sao Paulo state,Brazil

Hepatozoon canis is a protozoan that infects dogs and is transmitted by the ingestion of the brown dog tick, Rhipicephalus sanguineus. Two distinct species of Hepatozoon genus can infect dogs, H. canis and H. americanum. Routine tests to detect the disease are based on direct examination of gametocytes on Giemsa-stained blood smears. The objectives of this study were the investigation of infection prevalence in rural area dogs, the comparison of diagnostics by blood smear examination and polymerase chain reaction (PCR), and the association of infection with tick infestation. Blood smears, collected by puncture of the cephalic vein and ear margin capillary bed from 150 dogs, were examined. This technique detected 17 positive animals (11.3%), with 14 (9.3%) in peripheral blood and seven (4.7%) in cephalic vein blood. PCR tests detected 80 (53.3%) positive animals. R. sanguineus and Amblyomma spp. were found in 36 of the dogs (24%), in equal proportions. The identified species for Amblyomma genus were A. cajennense and A. ovale. Data analysis showed that PCR was much more sensitive when compared to blood smear examination. Hepatozoon species was previously identified as closely related to H. canis.     

Die Rhipicephalusarten der USSR,ein Beitrag zur Variabilität in der Sanguineusgruppe

Zusammenfassung Im Gebiet der USSR sind bisher die folgenden Vertreter der GattungRhipicephalus Koch nachgewiesen:Rhip. sanguineus sanguineus Latr.,Rhip. sanguineus rossicus Yak.,Rhip. schulzei Olen. undRhip. bursa Can. undFanz. Die subspezifische Stellung desrossicus wird begründet undpumilio P.Schulze (1935) als Synonym vonschulzei eingezogen.     

Molecular characterization and phylogeny of whipworm nematodes inferred from DNA sequences of cox1 mtDNA and 18S rDNA

A molecular phylogenetic hypothesis is presented for the genus Trichuris based on sequence data from the mitochondrial cytochrome c oxidase 1 (cox1) and ribosomal 18S genes. The taxa consisted of different described species and several host-associated isolates (undescribed taxa) of Trichuris collected from hosts from Spain. Sequence data from mitochondrial cox1 (partial gene) and nuclear 18S near-complete gene were analyzed by maximum likelihood and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. Phylogenetic results based on 18S ribosomal DNA (rDNA) were robust for relationships among species; cox1 sequences delimited species and revealed phylogeographic variation, but most relationships among Trichuris species were poorly resolved by mitochondrial sequences. The phylogenetic hypotheses for both genes strongly supported monophyly of Trichuris, and distinct genetic lineages corresponding to described species or nematodes associated with certain hosts were recognized based on cox1 sequences. Phylogenetic reconstructions based on concatenated sequences of the two loci, cox1 (mitochondrial DNA (mtDNA)) and 18S rDNA, were congruent with the overall topology inferred from 18S and previously published results based on internal transcribed spacer sequences. Our results demonstrate that the 18S rDNA and cox1 mtDNA genes provide resolution at different levels, but together resolve relationships among geographic populations and species in the genus Trichuris.     

Efficacy of deltamethrin (Butox® 7.5 pour on) against nymphs and adults of ticks (Ixodes ricinus, Rhipicephalus sanguineus) in treated hair of cattle and sheep

Ticks are known to be able to transmit a broad spectrum of agents of diseases in cattle or sheep. Therefore, measurements are needed to keep ticks away from the body of any ruminant belonging to the agricultural life stock. The present study dealt with investigations to measure the efficacy of the insecticide deltamethrin (Butox? 7.5 pour on) against specimens of two important species (Ixodes ricinus and Rhipicephalus sanguineus). Four sheep and four young cattle were treated lege arte along the vertebral column with 10 ml Butox? (deltamethrin) per sheep or 30 ml Butox? per cattle. Day 7, 14, 21, and 28 after the treatment, hair was shaved off from the head, ears, the back, belly, and the feet being collected in separate, suitable plastic bags, and transported to the institute, where these hair were brought into close contact with either adult and/or nymph stages of I. ricinus and R. sanguineus. As results, strong, acaricidal effects were seen, which varied according to the parasite species, the origin of the hair (e.g., head, leg, etc.) and according to the period after the treatment. In sheep, the acaricidal effect was noted for the whole period of 28 days along the whole body with respect to adults and nymphs of I. ricinus, while the acaricidal effects of deltamethrin were reduced for R. sanguineus stages beginning at day 21 after treatment. In cattle, the full acaricidal effect was seen for 21 days in I. ricinus stages and for 14 days in R. sanguineus, while the acaricidal efficacy became reduced after these periods of full action—beginning at the hair taken from the legs. Only R. sanguineus adults did not show any reaction on day 28 after treatment. Besides these acaricidal effects, repellent effects were also noted. Full repellency for both species was seen during the first 14 days in sheep and cattle against Ixodes and Rhipicephalus, while the repellency was later reduced, especially in contact with hair from the legs. As conclusion, deltamethrin, besides its very good effects against biting insects, brings acaricidal as well as repellent effects against ticks, thus protecting the sheep and cattle from transmission of agents of diseases.     

Factors affecting patterns of tick parasitism on forest rodents in tick-borne encephalitis risk areas,Germany

Identifying factors affecting individual vector burdens is essential for understanding infectious disease systems. Drawing upon data of a rodent monitoring programme conducted in nine different forest patches in southern Hesse, Germany, we developed models which predict tick (Ixodes spp. and Dermacentor spp.) burdens on two rodent species Apodemus flavicollis and Myodes glareolus. Models for the two rodent species were broadly similar but differed in some aspects. Patterns of Ixodes spp. burdens were influenced by extrinsic factors such as season, unexplained spatial variation (both species), relative humidity and vegetation cover (A. flavicollis). We found support for the ‘body mass’ (tick burdens increase with body mass/age) and for the ‘dilution’ hypothesis (tick burdens decline with increasing rodent densities) and little support for the ‘sex-bias’ hypothesis (both species). Surprisingly, roe deer densities were not correlated with larvae counts on rodents. Factors influencing the mean burden did not significantly explain the observed dispersion of tick counts. Co-feeding aggregations, which are essential for tick-borne disease transmission, were mainly found in A. flavicollis of high body mass trapped in areas with fast increase in spring temperatures. Locally, Dermacentor spp. appears to be an important parasite on A. flavicollis and M. glareolus. Dermacentor spp. was rather confined to areas with higher average temperatures during the vegetation period. Nymphs of Dermacentor spp. mainly fed on M. glareolus and were seldom found on A. flavicollis. Whereas Ixodes spp. is the dominant tick genus in woodlands of our study area, the distribution and epidemiological role of Dermacentor spp. should be monitored closely.     

Modification of universal 12S rDNA primers for specific amplification of contaminated Taenia spp. (Cestoda) gDNA enabling phylogenetic studies

The construction of new specific tapeworm primers allowed synthesis of a 311-bp fragment of the mitochondrial 12S rDNA of 11 Taenia species and two Echinococcus species by PCR. After direct sequencing and construction of an alignment, the DNA sequences were calculated by three different phylogenetic algorithms. The phylogenetic trees were tested by 1000 bootstrap replications. Reliability of the nodes was tested by splits testing. All three algorithms revealed a clear monophyletic phylum Taenia, suggesting it may be paraphyletic with respect to the genus Echinococcus. Within the genus Taenia, the first secure group was composed by Taenia saginata, T. solium, T. serialis, T. ovis and T. hydatigena. A delimited second group was formed by T. martis, T. taeniaeformis, T. mustelae and T. parva. All of them were opposed to the genus Echinococcus using other cyclophyllideans as an outgroup. In this study Echinococcus was used as an outgroup, being the closest species against which the ingroup could be routed. The findings of this publication reflect Verster's basic morphologically based grouping of the Taeniidae. Received: 9 March 1999 / Accepted: 22 April 1999     

Conservation and immunogenicity of the mosquito ortholog of the tick-protective antigen, subolesin

The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. The ortholog of the tick-protective antigen, subolesin, was identified in Aedes albopictus and found to have conserved epitopes in ticks and mosquitoes. RNA interference with the tick and mosquito double-stranded RNA in three tick species resulted in significant gene knockdown and decreased tick weight and/or survival. Feeding Anopheles atroparvus, Aedes caspius, and Culex pipiens female mosquitoes on an A. albopictus subolesin hyperimmune serum resulted in 11 ± 5% to 29 ± 6% survival inhibition when compared to controls fed on preimmune serum. Feeding sand flies, Phlebotomus perniciosus, on antimosquito subolesin ortholog protein antibodies inhibited female survival and the number of larvae and adults obtained after hatching by 28 ± 22% and 16 ± 3%, respectively, when compared to controls. Vaccination with tick and mosquito subolesin ortholog proteins significantly reduced Ixodes scapularis tick infestation and weight in a similar way. However, vaccination with the recombinant mosquito subolesin ortholog antigen did not protect against Amblyomma americanum and Rhipicephalus sanguineus tick infestations. Collectively, these preliminary results provided the first evidence that development of vaccines may be possible for control of multiple arthropod vectors using subolesin orthologs but suggested that multiple antigens may be required to produce an effective vaccine.     

Molecular characterization of hard and soft ticks from Romania by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, four hard tick species and one soft tick species, namely, Dermacentor marginatus, Haemaphysalis punctata, Haemaphysalis parva, Ixodes ricinus, and Dermanyssus gallinae, from south-western Romania were characterized genetically by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA), using a hard tick, Haemaphysalis longicornis, from China for comparative purposes. The ITS rDNA was amplified by polymerase chain reaction (PCR) and sequenced from individual ticks. The lengths of the ITS-1 sequences were 238–1819 bp, and the lengths of ITS-2 were 137–1695 bp, respectively, for all ticks sequenced. While sequence variation within a hard tick species was 0–1.5%, nucleotide differences between hard tick species ranged 2–25.2%, indicating that ITS rDNA sequences provide genetic markers for the differentiation of hard ticks from Romania. Hence, a PCR-linked restriction fragment length polymorphism approach was developed for their unequivocal differentiation based on ITS-1 rDNA. This is the first characterization of ticks from Romania using a genetic approach, which provides the foundation for further studies on ticks in Romania and has implications for studying the population genetic structure of the Romanian ticks and for identification and differentiation of closely related ticks. An erratum to this article can be found at     

The radiation of Thaparocleidus (Monogenoidea: Dactylogyridae: Ancylodiscoidinae): phylogenetic analyses and taxonomic implications inferred from ribosomal DNA sequences

The monophyly of Thaparocleidus Jain 1952 and its phylogenetic relationship with Pseudancylodiscoides were assessed using the D1–D2 domains of the large-subunit ribosomal deoxyribonucleic acid sequences, and taxonomic implications were discussed concerning the relative taxonomic importance of the characters of the reproductive complex and those of the haptoral scleties for future genus erection and combination within the Ancylodiscoidinae. Thaparocleidus contained divergent genetic lineages and was not resolved as a monophyletic group. The first lineage was represented by two species found on Pangasium sutchi (Pangasidae), and the second one contained 12 species all from Silurus astus (Siluridae). Three clades were observed for species from S. astus, consistent with results of morphological analyses, indicating that 12 Thaparocleidus species could be divided into three groups by morphological examinations of the male copulatory organ (MCO). Pseudancylodiscoides spp. was more closely related to Thaparocleidus spp. from S. astus, and morphologically, it was found that shapes of MCOs of these species were the same type but different from that of Thaparocleidus spp. from P. sutchi. Therefore, based on results of molecular phylogenetic analyses and morphological examinations, we propose to abolish Pseudancylodiscoides and to erect a new genus to accommodate Thaparocleidus species from S. astus and Pseudancylodiscoides spp. It is also suggested to erect another new genus to accommodate two Thaparocleidus species from the P. sutchi, whose MCO shapes are different from other Thaparocleidus species.     

Complete mitochondrial genomes of the three brown algae (Heterokonta: Phaeophyceae) Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis

We report the complete mitochondrial sequences of three brown algae (Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis) belonging to three phaeophycean lineages. They have circular mapping organization and contain almost the same set of mitochondrial genes, despite their size differences (31,617, 36,392 and 39,049 bp, respectively). These include the genes for three rRNAs (23S, 16S and 5S), 25–26 tRNAs, 35 known mitochondrial proteins and 3–4 ORFs. This gene set complements two previously studied brown algal mtDNAs, Pylaiella littoralis and Laminaria digitata. Exceptions to the very similar overall organization include the displacement of orfs, tRNA genes and four protein-coding genes found at different locations in the D. dichotoma mitochondrial genome. We present a phylogenetic analysis based on ten concatenated genes (7,479 nucleotides) and 29 taxa. Stramenopiles were always monophyletic with heterotrophic species at the base. Results support both multiple primary and multiple secondary acquisitions of plastids. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers AY494079, AY500368 and AY500367.     

Genotypic status of Babesia microti within the piroplasms

For resolution of the controversial taxonomic status of Babesia microti in relation to other Babesia and Theileria spp. a phylogenetic analysis of an American and a German B. microti strain was performed on the basis of sequences of the small-subunit rRNA gene (rDNA) using distance-matrix, maximum-parsimony, and maximum-likelihood algorithms. Both B. microti isolates clearly separated from a group containing other Babesia spp. as well as from a second group consisting of Theileria spp. Interestingly, the B. microti isolates clustered in a monophyletic group together with other piroplasm species of unclear taxonomic status, B. rodhaini, and a recently described small canine piroplasm species. These results support the existence of a third taxonomic entity of equal rank besides the Babesiidae and Theileriidae. Received: 11 February 2000 / Accepted: 24 February 2000     

Detection of bacteria related to <Emphasis Type="Italic">Candidatus</Emphasis> Midichloria mitochondrii in tick cell lines

Many ticks have been shown to be infected with intracellular bacteria. One of these bacteria is Candidatus Midichloria mitochondrii which is the only characterized bacterium that has the ability to invade the mitochondria within ovarian cells and consume them without any effect on the female tick’s reproduction. In the present study, eight cell lines derived from the ticks Ixodes ricinus, Ixodes scapularis, Rhipicephalus (Boophilus) microplus, and Rhipicephalus (Boophilus) decoloratus were examined for the presence of the bacterium Ca. Midichloria mitochondrii. PCR assays for this bacterium were carried out using two sets of primers targeting the eubacterial 16SrRNA gene and a set of primers specific for the gyrB gene of Ca. Midichloria mitochondrii. With the 16S rRNA primers, DNA was amplified from two cell lines (R. (B.) decoloratus line BDE/CTVM14 and I. ricinus line IRE/CTVM19) on one out of three occasions each. Sequencing of the PCR products showed that the two cell lines gave sequences with 100% similarity to Ca. Midichloria mitochondrii. However, all cell lines, including the two positive cell lines, were negative with the specific primers. Phylogenetic analysis shows that our sequences belong to the subclass α-proteobacteria. They were identical to the sequences amplified from the tick I. ricinus. The results suggest that two cell lines, IRE/CTVM19 and BDE/CTVM14, may contain bacteria closely related to Ca. Midichloria mitochondrii and identical with it in a 350-bp part of the 16S rRNA gene sequence. To our knowledge, this constitutes the first report of the presence of DNA similar to the DNA of Ca. Midichloria mitochondrii in tick cell lines.     

Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis

The vectorial capacity of Rhipicephalus sanguineus in the transmission of canine visceral leishmaniasis has been evaluated through a laboratory-controlled experiment. One healthy Leishmania-free dog and two dogs naturally infected with Leishmania were infested with R. sanguineus in various stages of development. Engorged larvae, unfed nymphs, engorged nymphs, unfed adults, engorged female adults and fed male adults were collected from the experimental animals and examined for Leishmania infection by optical microscopy, polymerase chain reaction (PCR) and parasite culture. Leishmania forms were not detected in any of the 433 smears prepared from engorged colonies nor in any of the 118 smears prepared from unfed colonies. However, one flagellate structure was identified in one of the smears. All pools of R. sanguineus that had fed on the infected dogs tested PCR-positive for Leishmania DNA, with the single exception of the pool of engorged larvae. In contrast, all pools of ticks that had fed on the Leishmania-free dog were PCR-negative. Leishmania growth was not observed in any of the tick colonies following incubation on culture medium. Considering that no Leishmania forms were identified in any of the meticulously analysed smears derived from engorged colonies of R. sanguineus, it appears somewhat unlikely that the maintenance and multiplication of Leishmania occurs within the tick.     

Comparing chromosomal and mitochondrial phylogenies of the Indriidae (Primates,Lemuriformes)

The Malagasy primate family Indriidae comprises three genera with up to 19 species. Cytogenetic and molecular phylogenies of the Indriidae have been performed with special attention to the genus Propithecus. Comparative R-banding and FISH with human paints were applied to karyotypes of representatives of all three genera and confirmed most of the earlier R-banding results. However, additional chromosomal rearrangements were detected. A reticulated and a cladistic phylogeny, the latter including hemiplasies, have been performed. Cladistic analysis of cytogenetic data resulted in a phylogenetic tree revealing (1) monophyly of the family Indriidae, (2) monophyly of the genus Avahi, (3) sister–group relationships between Propithecus diadema and Propithecus edwardsi, and (4) the grouping of the latter with Indri indri, Propithecus verreauxi, and Propithecus tattersalli, and thus suggesting paraphyly of the genus Propithecus. A molecular phylogeny based on complete mitochondrial cytochrome b sequences of 16 species indicated some identical relationships, such as the monophyly of Avahi and the sister–group relationships of the eastern (P. diadema and P. edwardsi) to the western Propithecus species (P. verreauxi, Propithecus coquereli, and P. tattersalli). However, the main difference between the molecular and cytogenetic phylogenies consists in an early divergence of Indri in the molecular phylogeny while in the chromosomal phylogeny it is nested within Propithecus. The similarities and differences between molecular and cytogenetic phylogenies in relation to data on the species’ geographic distributions and mating systems allow us to propose a scenario of the evolution of Indriidae. Chromosomal and molecular processes alone or in combination created a reproductive barrier that was then followed by further speciation processes.