Cytotoxic effects of andiroba oil (Carapa guianensis) in reproductive system of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females

The present study performed an analysis about the effects of andiroba seed oil (Carapa guianensis) in the ovary of Rhipicephalus sanguineus semi-engorged females; once, there are few studies about the action of natural products on the reproductive system, a vital organ for the biological success of this animal group. The results showed that andiroba oil is a potent natural agent which causes significant structural changes in the oocytes, such as the emergence of large vacuolated cytoplasmic regions, reduction in the number of yolk granules, changes in the shape of the cells, as well as impairment of genetic material. In addition, the ovary epithelium showed severe morphological changes, such as extreme structural disorganization, with highly vacuolated cells and picnotic nuclei, forming an amorphous mass. This study showed also that oocytes (mainly in the initial stages of development) and the ovary epithelium of R. sanguineus females subjected to different concentrations of andiroba oil presented morphological changes which became more numerous and intense as the concentration of the product increased. Based on the results, it can be inferred that although the defense mechanisms are developed by oocytes to recover the cellular integrity (presence of autophagic vacuoles), these cells are not able to revert the damage caused by this product. Thus, it can be concluded that although the damages caused to the oocytes by andiroba oil are comparatively less severe than the ones caused by synthetic acaricides, this product can be considered a potent natural agent that reduce and/or prevent the reproduction of R. sanguineus females, with the advantage of not causing environmental impact such as synthetic chemical acaricides.     

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.     

Inoculation of salivary gland extracts obtained from female of Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae) with 2, 4, and 6 days of feeding in rabbit: I—histopathology of the feeding lesion

This study analyzed the histopathology of rabbit skin, previously immunized with SGE2, SGE4, and SGE6 gland extracts prepared from salivary glands of Rhipicephalus sanguineus female with 2, 4, and 6 days of feeding, at the region of the R. sanguineus female feeding lesion 2, 4, and 6 days after tick attachment. In this work, infestation-naïve New Zealand White rabbits were inoculated either with the extracts (test group (TG)) or with phosphate buffer and complete Freund’s adjuvant mixture (control group 2 (CG2)). Each extract-inoculated- (TG and CG2) and non-inoculated (CG1) rabbit was subsequently infested with R. sanguineus. Skin biopsies were collected from the rabbit at the tick feeding lesion at 2, 4, and 6 days of feeding. Results revealed that rabbit immunization with gland extracts induced acquisition of resistance against this species. It should be stated that the SGE4 extract was the most effective in developing an immune–inflammatory response against ectoparasites, being this process characterized by the presence of an early and intense inflammatory cell infiltrate. On the other hand, SGE6 extract caused a later appearance of resistance with less infiltrate occurrence and intense edema at the feeding lesion site. As to the inflammatory process deriving from SGE2 extract inoculation, it was the less intense. It was concluded that immunization with different extracts from R. sanguineus female salivary glands did not change microscope features of the inflammatory process, although an earlier or more intense and later response, which was also dependent on the inoculate extract, was noticed.     

The dog factor in brown dog tick Rhipicephalus sanguineus (Acari:Ixodidae) infestations in and near human dwellings

Three cases of the tick Rhipicephalus sanguineus infiltration in or near human dwellings caused by dogs, and their influence on epidemiological features of human habitats have been investigated, (a) The observation of dogs kept indoors proved that single tick females could engorge and oviposit inside apartments followed by the development of subadults. (b) Abundant micropopulations of ticks were formed in small yards or gardens near the dwellings where dogs lived in kennels, (c) A huge field population of R. sanguineus was observed on a farm where watchdogs constantly patrolled along the farm perimeter. Tick abundance near the kennels and in the permanent resting sites of the dogs reached more than 30 adults per 10 min of collecting, while the number of adults on a dog reached 100. Unfed adult females under conditions of constant dog availability had a larger scutal index than females collected in the control field site. On the basis of circumstantial evidence it is possible to conclude that under the above conditions tick development may change from the normal 3-host cycle to a 2-host cycle. Ticks in the field had one complete generation per year. Ticks on the farm, as well as ticks in kennels, developed faster and a significant part of their population had two complete generations per year. R. sanguineus is the main vector and reservoir of a pathogen from the Rickettsia conorii complex, the causative agent of Israeli tick typhus. The described conglomerations of R. sanguineus create a great risk to humans who can be attacked by infected ticks in and around their homes, even in large towns. Such a feature of the tick life history most likely exists not only in Israel but in other countries as well.     

Ticks’ response to feeding on host immunized with glandular extracts of <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> females fed for 2, 4, and 6 days. I. Inactivity or early degeneration of salivary glands?

The present study histologically analyzed the salivary glands of Rhipicephalus sanguineus females fed for 2, 4, and 6 days in hosts which had been previously immunized with glandular extracts obtained from females from this same species in different periods of feeding, having as main objective verify the action of these extracts in the secretor cycle of these glands. For this, glandular extract of females fed for 2 days (SGE2), glandular extract of females fed for 4 days (SGE4), and glandular extract of females fed for 6 days (SGE6) extracts were obtained from salivary glands of R. sanguineus females fed for 2, 4, and 6 days respectively. Then, New Zealand White naive rabbits were inoculated either with extracts (test group = TG), or with a mixture of phosphate buffer and Freund's complete adjuvant (control group 2 = CG2). Each inoculated rabbit (TG and CG2) and non-inoculated (control group 1 = CG1) was posteriorly infested with 15 couples of fasting R. sanguineus from which the salivary glands had been collected from females fed for 2, 4, and 6 days. The results revealed that the resistance the hosts had acquired by the immunization with the extracts affected differently the secretory activity of the glandular cells. It was verified that the resistance to SGE2 and SGE4 extracts acted in the cells of acini II and III, being c1 and c5 from II and d from III inactivated due to the action of SGE2 and c1 and c4 from II and f from III inactivated by the action of SGE4. As for the resistance to SGE6 the effect was only on cells of acini II (c1, c3 e c4), which were also inactivated. In addition, the hosts’ resistance to SGE2–SGE6 extracts made the degenerative process earlier in comparison to CG1. On the other hand, the resistance to the extracts did not influence the characteristics of the degenerative process normally found in salivary glands. The assynchronism of the degenerative process was maintained—acini III were always the most affected and I the less affected. The structural cell alterations, such as cytoplasmic vacuolation, nuclear alterations and formation of apoptotic bodies which characterize the occurrence of atypical apoptosis were also maintained in the glands of individuals from TG making it clear that the immunization of the hosts with glandular extracts SGE2–SGE6 had influenced the glandular physiology of R. sanguineus, which is an important piece of information in the search for a way to control these ectoparasites.     

Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae)

The mitochondrial 16S rRNA gene sequences of the following eight European Metastriata tick species were obtained by direct polymerase-chain-reaction cycle sequencing and silver-staining methods: Rhipicephalus bursa, R. pusillus, R. sanguineus, R. turanicus, Boophilus annulatus, Dermacentor marginatus, Haemaphysalis punctata, and Hyalomma lusitanicum. This mitochondrial gene seems to be a good marker for the establishment of genetic relationships among closely related tick species, but it does not seem to be useful for comparisons of distantly related taxa. The molecular data provide very strong support for the monophyly of the Rhipicephalinae, including Hyalomma spp. However, the genus Rhipicephalus may not be considered a monophyletic group; in all analyses carried out in this study, R. bursa clustered with Boophilus spp. The high percentage of similarity (98.7%) observed between R.␣sanguineus and R. turanicus sequences would suggest that these species recently diverged within the Rhipicephalus genus. Phylogenetic analyses showed a monophyletic relationship among Amblyomminae taxa. The relationships between Haemaphysalis species and the true placement of this genus within Metastriata could not be resolved. Received: 12 September 1997 / Accepted: 3 December 1997     

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.     

Rhipicephalus sanguineus (Acari: Ixodidae) female ticks exposed to castor oil (Ricinus communis): an ultrastructural overview

Tick control has been accomplished through the use of synthetic acaricides, which has created resistant individuals, as well as contaminating the environment and nontarget organisms. Substances of plant origin, such as oils and extracts of eucalyptus and neem leaves, have been researched as an alternative to replace the synthetic acaricides. Ricinoleic acid esters from castor oil have recently been shown as a promising alternative in eliminating bacterial contamination during ethanol fermentation, by acting as an effective biocide. The same positive results have been observed when these esters are added to the food given to tick-infested rabbits. This study tested the effect of these substance on the reproductive system of Rhipicephalus sanguineus females, added to rabbit food, more specifically on oogenesis. For this, four groups were established: four control groups (CG1, CG2, CG3, and CG4) and four treatment groups (TG1, TG2, TG3, and TG4) with one rabbit in each (New Zealand White), used as hosts. After full 4 days feeding (semi-engorgement), the females were collected and had their ovaries extracted. In this study, it was observed that R. sanguineus females exposed to esters had their ovaries modified, which was demonstrated through transmission electron microscopy techniques. The addition of ricinoleic esters to the diet of tick-infested rabbits revealed how toxic such substances are for the cytoplasmic organelles of oocytes and pedicel cells. These compounds can change the morphophysiology of germ and somatic cells, consequently influencing their viability and, therefore, confirming that the ricinoleic acid esters from castor oil are a promising substance in the control of R. sanguineus.     

Morphology of the midgut of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) adult ticks in different feeding stages

The intestinal epithelial cells of ticks are fundamental for their full feeding and reproductive success, besides being considered important sites for the development of pathogens. Rhipicephalus sanguineus ticks are known for their great medical and veterinary importance, and for this reason, the knowledge of their intestinal morphology may provide relevant subsidies for the control of these animals, either by direct acaricidal action over these cells or by the production of vaccines. Therefore, this study aimed to describe the midgut morphology of male and female R. sanguineus ticks in different feeding stages, by means of histological analysis. Significant differences were observed between the genders, and such alterations may refer mainly to the distinct demands for nutrients, much higher in females, which need to develop and carry out the egg-laying process. In general, the midgut is coated by a thin muscle layer and presents a pseudostratified epithelium, in which two basic types of cells can be observed, connected to a basal membrane—generative or stem and digestive cells. The latter was classified as follows: residual, deriving from the phase anterior to ecdysis; pinocytic, with vesicles containing liquid or pre-digested components of blood; phagocytic, with entire cells or remnants of nuclear material inside cytoplasmic vesicles; and mature, free in the lumen. Digestion is presumably intracellular and asynchronous and corresponds to a process which starts with the differentiation of generative cells into pinocytic digestive cells, which subsequently start to phagocytize intact blood cells and finally detach from the epithelium, being eliminated with feces.     

Evaluation of the acaricide activity of thymol on engorged and unengorged larvae of Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae)

The present work was aimed at evaluating the in vitro efficacy of thymol on engorged and unengorged larvae of Rhipicephalus sanguineus. In order to perform the study, unengorged larvae were interleaved on filter paper (2 × 2 cm) recently sprayed with thymol in the following concentrations: 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%. For each concentration, ten repetitions of 50 larvae each were performed. A control group was established (water + DMSO) together with a positive control group (Amitraz), with ten repetitions each. Mortality was evaluated after 24 h. For the test with engorged larvae, the same thymol concentrations were used with the larvae being immersed for 5 min and mortality evaluated after 15 days. The values found for thymol efficacy for unengorged larvae were of 21.3%, 17.4%, 19.1%, 32.1%, 37.7% and for engorged larvae 3.0%, 0.0%, 3.0%, 97.0%, and 100.0% in concentrations of 0.25%, 0.5%, 1.0%, 1.5%, and 2.0% respectively. We have concluded that thymol had a deleterious effect on larvae of R. sanguineus, especially on engorged larvae for which 100% mortality was found in higher concentration.     

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.     

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.     

Zur Ökologie eines deutschen Stammes von Rhipicephalus sanguineus (Latreille, 1806)

Zusammenfassung Zur experimentellen Bewertung der ökologischen Toleranzkapazität von Rhipicephalus sanguineus wurden alle postembryonalen Entwicklungsstadien dieser Zeckenart im nüchternen und vollgesaugten Zustand verschiedenen Kombinationen von Temperatur- und relativen Luftfeuchtigkeitsbedingungen ausgesetzt, um den Einfluß dieser beiden abiotischen Faktoren auf ihre Entwicklung, Reproduktionskapazität sowie ihr Überleben zu untersuchen.In Untersuchungen zur ökologischen Reaktionsbreite von Rhipicephalus sanguineus konnte eindeutig nachgewiesen werden, daß die existentielle Sicherung dieser Zeckenart sowie deren Entwicklungs- und Reproduktionsvermögen von Temperatur und relativer Luftfeuchtigkeit bestimmt werden. Diese Beeinflussung ist dabei allgemein durch eine direkt proportionale Abhängigkeit von der relativen Luftfeuchtigkeit und eine umgekehrt proportionale Relation zur Temperatur gekennzeichnet.Das Propagationsvermögen zur enzootischen Manifestation ist nur im engen Temperaturbereich von 20–30° C und bei hoher relativer Luftfeuchtigkeit möglich. Diese Zeckenart ist jedoch in allen ihren Entwicklungsstadien für eine bestimmte Zeit fähig, selbst kritische Temperatur- und Feuchtigkeitsbedingungen zu tolerieren und damit ihren enzootischen Fortbestand zu sichern. Rhipicephalus sanguineus kann zwar auf Grund kritischer Makroklimabedingungen als Freilandzecke in Deutschland nicht bestehen, durch ihre besondere Wirtsprädilektion für Hunde wird dieser Zeckenart jedoch ein Biotop geboten, in dem alle ökologischen Voraussetzungen zur autochthon enzootischen Manifestation und epizootischen Ausbreitung gegeben sind.

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On the Ecology of a German Strain of Rhipicephalus sanguineus (Latreille, 1806)

Summary Experimental evaluation of the ecological tolerance of Rhipicephalus sanguineus was carried out by exposing all the postembryonic stages of development of this tick species to various combinations of temperature and relative humidity conditions, in both its fasting and replete phases, in order to investigate the influence of these two abiotic factors on their development and reproductive capacity, as well as on their ability to survive.Experiments into the degree of reaction to its environment showed unequivocally that the security of survival of Rhipicephalus sanguineus, as well its capacity for development and reproduction, are determined by temperature and relative humidity. These influences are characterized by a generally directly proportional relationship to relative humidity and an indirectly proportional relationship to temperature.The ability to propagate to the enzootic manifestation is only possible within the restricted range of temperature of 20–30° C and at a relatively high humidity. This tick is however capable of enduring critical temperature and humidity conditions for a given length of time, thus ensuring the continuation of the enzootic state. Rhipicephalus sanguineus is however unable to exist as a field tick due to critical macroclimatic conditions in Germany in its special predilection for the dog as a host this species is presented with a biotope in which all the ecological prerequisites are available for its autochthonous enzootic manifestation and its epizootic dissemination.

     

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.     

Spotted fever group <Emphasis Type="Italic">Rickettsia</Emphasis> in brown dog ticks <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> in southwestern Spain

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.     

Ultrastructural analysis of the oocytes of female Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks subjected to the action of Azadirachta indica A. Juss (neem)

The present study provides ultrastructural information about the acaricidal effects of neem (Azadirachta indica A. Juss) on the ovaries of R. sanguineus engorged females. In general, the main damage caused in the oocytes was alteration in the shape of the cell and of the germinal vesicle, ring-shaped nucleolus, cytoplasmic vacuolation, and disorganization of the organelles and of the cell membranes (including the chorion), all of which indicate that these cells could be in the process of death. The results showed that azadirachtin would be an efficient acaricide agent for inhibiting and/or neutralizing the reproduction process of R. sanguineus females, impairing the reproductive ability of this species.     

Acaricidal efficacy of thymol on engorged nymphs and females of Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae)

The present work was aimed at evaluating the “in vitro” efficacy of different concentrations of thymol on engorged nymphs and females of Rhipicephalus sanguineus. The nymphs were separated in seven groups and immersed in different concentrations of thymol (0.25%, 0.5%, 1.0%, 1.5%, and 2.0%) for 5 min. A control group was established (water + dimethylsulfoxide) together with a positive control group (Amitraz*), and mortality was evaluated after 15 days. In order to perform the experiment with females, the same methodology was used, and the following concentrations were tested: 1.0%, 1.5% and 2.0%. After 5 min of immersion, the engorged females were fixed on Petri dishes with the aid of a double-sided adhesive tape and kept in a climatized room regulated at 27 ± 1°C and UR >80 ± 10%, and different parameters referring to the biology of the non-parasitary phase were evaluated daily. The values found for thymol efficacy on nymphs were 0.0%, 100%, 100%, 100%, and 100% in concentrations of 0.25%, 0.5%, 1.0%, 1.5%, and 2.0%, respectively. In the experiment with engorged females, thymol did not induce any significant alterations (p < 0.05) in the parameters of weight alteration, egg mass weight, pre-oviposition period, hatching percentage, egg production index and nutritional index; however, it affected the engorged females final weight in all treatments (p > 0.05). The concentration of thymol 2% was the one that showed a better efficacy (41%). It was concluded that thymol had a more accentuated deleterious effect on engorged nymphs, and it might be a promising supporting agent for the control of this ixodid.     

Cell Death in Salivary Glands of Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) Females at Semi-engorged Feeding Stage

The ultrastructure of the salivary glands of Rhipicephalus (Boophilus) microplus females is described during feeding. In beginning of feeding, individuals show acini I with many mitochondria and wide basal labyrinth in peripheral cells; glycoprotein granules only in b and c₃ cells (acini II); and epithelial interstitial cells with developed basal labyrinth between f cells (acini III). Semi-engorged females show cells in degeneration, with autophagic vacuoles, lysosomes, myelin figures, and irregular, condensed, and/or fragmented nuclei, in addition to apoptotic bodies. R. B. microplus points to apoptosis in these organs before the detachment from the host, in contrast to others tick species.     

Identification of hard ticks collected from sheep naturally infected with Anaplasma ovis in Isfahan province, central Iran

Attached ticks and blood samples were collected from 150 sheep in Isfahan province, central part of Iran. Blood samples were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Of the 150 sheep examined, 50 (33.33%) were found positive for Anaplasma ovis by PCR-RFLP. Of 50 sheep naturally infected with A. ovis, 553 ixodid ticks were collected. The ticks were identified (three species belonging to two genera) as follows: Rhipicephalus sanguineus (53.9%), Hyalomma marginatum marginatum (27.5%), and Hyalomma anatolicum anatolicum (18.6%). All A. ovis-infected sheep were infested with R. sanguineus. This suggests that R. sanguineus may be one of the main vectors of sheep anaplasmosis in the central part of Iran.     

Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy

Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.