Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6).

Iron is a very important nutrient for cells; however, it could also cause fatal effects because of its capability to trigger oxidative stress. Due to high exposure to iron from their blood diet, ticks make use of several mechanisms to cope up with oxidative stress. One mechanism is iron sequestration by ferritin and its control protein (IRP). Since the IRP activity is dependent on the ferrous iron concentration, we tried to induce intracellular ferritin (FER1) protein expression by exposing Ixodes scapularis embryo-derived cell line (ISE6) to different concentrations of ferrous sulphate at different time points. We were able to induce FER1 protein after exposure to 2 mM of ferrous sulphate for 48 h, as observed in both Western blotting and indirect immunofluorescent antibody tests. This could indicate that the FER1 produced could be a product of the release of IRPs from the FER1 mRNA leading to its translation. The RNA interference of FER1, through the transfection of dsRNA, led to an increase in mortality and decrease in the cellular proliferation of ISE6 cells. Overall, ISE6 cells could be a good tool in further understanding the mechanism of FER1 action, not just in Ixodes ticks but in other tick species as well.

Infection of Ixodes spp. tick cells with different Anaplasma phagocytophilum isolates induces the inhibition of apoptotic cell death.

Anaplasma phagocytophilum is an intracellular rickettsial pathogen transmitted by Ixodes spp. ticks, which causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever (TBF) in ruminants. In the United States, human granulocytic anaplasmosis (HGA) is highly prevalent while TBF has not been reported. However, in Europe the situation is the opposite, with high prevalence for TBF in sheep and low prevalence of HGA. The origin of these differences has not been identified and our hypothesis is that different A. phagocytophilum isolates impact differently on tick vector capacity through inhibition of apoptosis to establish infection of the tick vector. In this study we used three different isolates of A. phagocytophilum of human, canine and ovine origin to infect the Ixodes ricinus-derived cell line IRE/CTVM20 and the Ixodes scapularis-derived cell line ISE6 in order to characterize the effect of infection on the level of tick cell apoptosis. Inhibition of apoptosis was observed by flow cytometry as early as 24h post-infection for both tick cell lines and all three isolates of A. phagocytophilum, suggesting that pathogen infection inhibits apoptotic pathways to facilitate infection independently of the origin of the A. phagocytophilum isolate and tick vector species. However, infection with A. phagocytophilum isolates inhibited the intrinsic apoptosis pathway at different levels in I. scapularis and I. ricinus cells. These results suggested an impact of vector-pathogen co-evolution on the adaptation of A. phagocytophilum isolates to grow in tick cells as each isolate grew better in the tick cell line derived from its natural vector species. These results increase our understanding of the mechanisms of A. phagocytophilum infection and multiplication and suggest that multiple mechanisms may affect disease prevalence in different geographical regions.

Ixodes pacificus ticks maintain embryogenesis and egg hatching after antibiotic treatment of Rickettsia endosymbiont.

Rickettsia is a genus of intracellular bacteria that causes a variety of diseases in humans and other mammals and associates with a diverse group of arthropods. Although Rickettsia appears to be common in ticks, most Rickettsia-tick relationships remain generally uncharacterized. The most intimate of these associations is Rickettsia species phylotype G021, a maternally and transstadially transmitted endosymbiont that resides in 100% of I. pacificus in California. We investigated the effects of this Rickettsia phylotype on I. pacificus reproductive fitness using selective antibiotic treatment. Ciprofloxacin was 10-fold more effective than tetracycline in eliminating Rickettsia from I. pacificus, and quantitative PCR results showed that eggs from the ciprofloxacin-treated ticks contained an average of 0.02 Rickettsia per egg cell as opposed to the average of 0.2 in the tetracycline-treated ticks. Ampicillin did not significantly affect the number of Rickettsia per tick cell in adults or eggs compared to the water-injected control ticks. We found no relationship between tick embryogenesis and rickettsial density in engorged I. pacificus females. Tetracycline treatment significantly delayed oviposition of I. pacificus ticks, but the antibiotic's effect was unlikely related to Rickettsia. We also demonstrated that Rickettsia-free eggs could successfully develop into larvae without any significant decrease in hatching compared to eggs containing Rickettsia. No significant differences in the incubation period, egg hatching rate, and the number of larvae were found between any of the antibiotic-treated groups and the water-injected tick control. We concluded that Rickettsia species phylotype G021 does not have an apparent effect on embryogenesis, oviposition, and egg hatching of I. pacificus.

Babesia bigemina: in vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells.

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.

Origin of alimentary tract in embryogenesis of Ixodes ricinus (Acari: Ixodidae).

We studied the embryos of Ixodes ricinus (L.) in the second and third trimester of embryonic development, by using light and transmission electron microscopy. At the beginning of the second trimester, the formation of the foregut and rectal sac, by a process of invagination, was observed. The invagination, which develops into the primordium of the hindgut, forms only in the third trimester. The rectum forms in the last phase of embryogenesis. The development of the midgut is incomplete during embryogenesis. The yolk is surrounded by a wall, formed of an amorphous basal lamina and flattened cells, that gradually accumulate deutoplasmic material. These cells do not acquire the typical features of the gut epithelium until after larval hatching. These features are, however, found in the cells forming the rectal sac.

Immunization of cattle with Anaplasma marginale derived from tick cell culture.

Anaplasmosis is a hemolytic disease of cattle caused by the ehrlichial tick-borne pathogen Anaplasma marginale. Killed vaccines used for control of anaplasmosis in the US used antigen harvested from infected bovine erythrocytes which was often contaminated with bovine cells and other pathogens. In this study, we performed an initial cattle trial to test A. marginale harvested from tick cell culture as an immunogen for cattle. Eleven yearling Holstein cattle were immunized with the cell culture-derived A. marginale and 11 cattle were non-immunized contact controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale in an oil-based adjuvant. Two immunizations were administered subcutaneously 4 weeks apart and the cattle were challenge-exposed 10 weeks after the second immunization with A. marginale infected blood. Maximum antibody levels as determined by an A. marginale specific competitive ELISA were observed 2 weeks after the last immunization. Antibody responses against major surface proteins (MSPs) 1a and 1beta1 were also characterized and immunized cattle demonstrated a preferential recognition for MSP1beta1. Cattle immunized with the cell culture-derived A. marginale had a significantly lower percent reduction in the packed cell volume (P<0.05) after challenge exposure as compared with the controls and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.

Morphology and development of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in cultured Ixodes scapularis (Acari: Ixodidae) cells.

Anaplasma marginale Theiler, a tick-borne rickettsial pathogen of cattle, was recently propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis Say. Cell monolayers were infected briefly with a high multiplicity of infection to synchronize rickettsial development and allow for description of the invasion, development, and release of A. marginale from the cultured cells. Sequential samples were collected, fixed, and processed for examination with light and electron microscopy. A. marginale entered host cells by an endocytotic process and remained within a vacuolar membrane throughout development. After entry, the dense form of A. marginale transformed into the vegetative or reticulated form that multiplied by binary fission, forming large colonies of rickettsiae. The reticulated form subsequently transformed into the dense form of A. marginale, which was released from cells and survived extracellularly. The dense forms were eventually released from the cultured cells by a process in which the inclusion membrane fused with the host cell membrane. Release of A. marginale was effected without the loss of host cell cytoplasm. In subsequent cell cycles, A. marginale reinfected cultured cells resulting in the development of multiple colonies per cell and eventual host cell destruction. Small vesicles were abundant within the colonies and appeared to form from individual rickettsiae. Development of A. marginale in IDE8 cells was similar to that described in naturally infected Dermacentor spp. ticks. However, destruction of cells by A. marginale as seen in vitro was not observed in naturally infected ticks. An understanding of the developmental cycle of A. marginale in cultured cells may provide insight into rickettsial development in its tick host and provide a basis for studying pathogen-host cell interaction in vitro.

Host specificity of Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) with comments on the drop-off rhythm

The parasitic specificity of larval, nymph and adult Amblyomma cajennense on six different host species: Oryctolagus cuniculus, Rattus norvegicus, Gallus gallus domesticus, Anas platyrhynchus, Coturnix coturnix and Streptopelia decorata is described. In terms of the numbers of larvae and nymphs recovered, O. cuniculus was the best host species. The modal day for drop-off of lavae and nymphs was day three for the mammal hosts, but variable in the birds. We conclude that adult A. cajennense have a strong degree of specificity due to the fact that the tick failed to complete its cycle on any of the evaluated hosts. The immature stages, on the other hand, showed a low level of specificity, most specially in the larval stage, indicating the existence of secondary hosts which probably serve as dispersers in the wild. The results also indicated a variable drop-off rhythm for larvae and numphs in two periods, diurnal (6-18 hr) and nocturnal (18-6 hr), which differed depending upon the host.

Study of the weight of eggs from six ixodid species from Brazil

Eggs from engorged females of six ixodidae species (Ixodes Iocatus, Amblyomma rotundatum, A. cajennense, Haemaphysalis leporis-palustris, Boophilus microplus, Dermacentor nitens) maintained in laboratory were counted to calculate the number and mean weight in Ig of eggs from each species. Phylogenetic considerations are discussed based on the results.

[Distribution of hungry and engorged Ixodes persulcatus (Ixodidae) imagos on squares for absolute enumeration]. / Raspredelenie golodnykh i sytykh imago taezhnogo kleshcha (Ixodidae) na ploshchadkakh absoliutnogo ucheta chislennosti.

It was found out by the methods of large scale cartography and mathematics modelling, that the distribution of Ixodes persulcatus ticks around the point of clutch is still aggregated both on stages of hungry larvae and nymphs and on a stage of hungry imago. It is proposed to use the number of hungry imago aggregates as the criterium for the estimation of clatch number per square unit. Wild animals living in foothill taiga forests of the central part of Krasnoyarsk region support 9-10 female ticks per 10,000 square meters, and among them 5-6 females rise in full-bodied offspring, which completes the life cycle.