Japanese Babesia microti cytologically detected in salivary glands of naturally infected tick Ixodes ovatus

Babesia microti protozoa were detected by light and electron microscopy in the salivary glands of field-collected Ixodes ovatus ticks; 6 of 85 adult ticks were demonstrated to be positive for B. microti DNA by polymerase chain reaction assays. In the salivary glands of unfed ticks, B. microti existed in the sporoblast stage in the granular acinus cells, and developed into the sporozoite stage during feeding on the host for 2 days. The present results indicated for the first time that I. ovatus can indeed carry B. microti and is not infected mechanically with the parasites by blood-sucking. This frequent infection of I. ovatus with B. microti demonstrates the significance of such a vector-pathogen relationship in Japan, and strongly suggests that I. ovatus is involved in the maintenance of B. microti in the fauna of Japanese rodents.     

Survival of Theileria parva-infected adult Rhipicephalus appendiculatus under laboratory and quasi-natural conditions

Adult Rhipicephalus appendiculatus Muguga, having high or low intensities of Theileria parva Muguga infection in their salivary glands, were exposed to 20 °C and 85% relative humidity in the laboratory or quasi-natural conditions. Survival of the ticks and T. parva infections in their salivary glands was then monitored over a two year period. Ticks, having an average infection level of 2 infected acini per female, survived for up to 70 or 106 weeks after moulting under the laboratory or quasi-natural conditions respectively. Those having an infection level of 26 infected acini per female, survived for a similar duration except that those under quasi-natural conditions survived for a slightly shorter duration (102 weeks). Similarly, T. parva parasites survived for much longer periods under quasi-natural conditions than under the laboratory conditions. They survived for up to 38 or 78 weeks post salivary gland infection under the laboratory or quasi-natural conditions respectively in both categories of infection levels. There was apparently a density dependent relationship in T. parva survival, with a dramatic fall in infection occurring in ticks with high levels of infection between weeks 10 and 18 or weeks 38 and 46 post salivary gland infection in those exposed to laboratory or quasi-natural conditions before levelling off. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.     

Susceptibility of Amblyomma americanum to natural and experimental infections with Theileria cervi

One hundred fifty Amblyomma americanum were examined between March and September 1986 from Cookson Hills Wildlife Refuge in eastern Oklahoma (USA). Of these ticks, 11% (17 of 150) were infected with Theileria cervi. Field-collected nymphal ticks had an 8% (3 of 37) prevalence of infection averaging 1.0 infected acini/nymph. Female ticks had a 16% prevalence of infection averaging 1.6 infected acini/female; T. cervi was not observed in salivary glands of field-collected male ticks. When laboratory reared A. americanum nymphs were allowed to feed on experimental white-tailed deer (Odocoileus virginianus) with varying T. cervi parasitemias (less than 1, 2, 6 and greater than 20%), only ticks which fed on deer with parasitemias greater than 1% became infected. Although prevalence and intensity of infection varied in the infected ticks, there was no significant difference in prevalence of infection between males and females. However, females did acquire significantly greater intensities than males. The data from these studies confirm that T. cervi overwinters in A. americanum and suggests that the prevalence, intensity and abundance of infection of T. cervi in ticks is influenced by the parasitemia of the deer host. Furthermore, fawns may play a more important role in the epidemiology of T. cervi transmission than do adult deer because of the coordination between tick activity patterns and deer fawning.     

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

Absence of spirochaetes (Borrelia burgdorferi) and piroplasms (Babesia microti) in deer ticks (Ixodes dammini) parasitized by chalcid wasps (Hunterellus hookeri)

An entomophagous wasp (Hunterellus hookeri Howard) parasitizes about a third of the host-seeking nymphal Ixodes dammini Spielman et al. ticks on Naushon Island in Massachusetts (U.S.A.) where the agents of Lyme disease (Borrelia burgdorferi Johnson et al.) and human babesiosis (Babesia microti Franca) are enzootic. Following blood-feeding, wasp-parasitized ticks are destroyed by the developing wasp. The prevalence of either human pathogen in host-seeking ticks collected in wasp-infested sites is nearly 40% lower than that found in other sites. Nymphal ticks, collected early in their season of activity, are more frequently parasitized by the wasp and less frequently by the Lyme disease spirochaete than those collected later in the summer. Spirochaetes never infected wasp-infected ticks, and few wasp-infected ticks were concurrently infected by the Babesia piroplasm. Taken together, these correlations indicate that the wasp may render the tick inhospitable to both pathogens. The presence of the wasp may have reduced risk of human infection on the island by either pathogen by as much as a third.     

Observations on the Development of Babesia caballi (Nuttall) in the Tropical Horse Tick Dermacentor nitens Neumann

SYNOPSIS. The development of Babesia caballi (Nuttall) in Dermacentor nitens Neumann was studied in smear preparations and histologic sections of ticks infected with this protozoan parasite. A majority of the parasites in equine erythrocytes ingested by the adult ticks apparently were destroyed. Smal spherical bodies 4–6 μ in diameter were the 1st developmental stages of B. caballi observed in the gut contents of ticks infected with this parasite. These spherical bodies apparently gave rise to clavate (club-shaped) bodies 10–14 μ long by 4–6 μ wide. The latter developed into large round bodies 12–16 μ in diameter that segmented into vermicular-shaped parasites, about 8–12 μ long by 2–4 μ wide; some penetrated the gut wall, some invaded other cells of the tick.
In the cells of the Malpighian tubules, hemolymph, and ovaries, the vermicular parasites underwent a secondary cycle of multiple fission, forming vermicules similar to those occurring earlier in the gut. Vermicules that invaded the ova underwent a similar multiple fission cycle during the larval stage of the tick.
Vermicules from the multiple fission cycle that occurred during the period of larval feeding invaded the salivary glands. A multiple fission cycle of increase within these glands resulted in large numbers of small, oval and piriform parasites, 2.5–3 μ, maximum dimension. These parasites became mixed with the salivary secretions, and presumably are the forms injected into the horse by the nymphs as they feed. The small oval and piriform parasites therefore appear to be the infective stage for the horse.     

Enzootic Babesia microti in Maine

Human babesiosis in the northeastern United States caused by Babesia microti (Apicomplexa: Piroplasmida) is mainly reported from coastal New England sites, where deer ticks (Ixodes dammini) are common. However, the piroplasm has been detected in microtine rodents elsewhere in association with I. angustus or other nidicolous ticks, suggesting that the agent is widely distributed but zoonotically significant only where a human-biting "bridge" vector is present. To determine whether this piroplasm may be enzootic in areas where I. dammini is absent, we surveyed small mammals collected from 2 sites in Maine, where I. angustus or I. muris is common but I. dammini is not. Of 43 chipmunks, voles, deer mice, and shrews examined, 3 (6.9, 95% confidence interval 0 to 14.5) were parasitemic, as determined by blood smear or polymerase chain reaction targeting a piroplasm-specific portion of the 18S ribosomal DNA gene. Phylogenetic analysis of the sequenced amplification products demonstrates the presence of 2 forms of B. microti. We conclude that B. microti may be enzootic in the absence of I. dammini but that human risk relates to dense infestations of this human-biting tick.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Transmission of Cytauxzoon felis by injection of Amblyomma americanum salivary glands

BackgroundCytauxzoonosis is a life-threatening disease of cats, caused by the tick-borne piroplasmid hemoparasite, Cytauxzoon felis. Current experimental models for cytauxzoonosis rely on either tick transmission or direct injection of infected cat tissues. These models require researchers to directly work with infected ticks or use cats with acute cytauxzoonosis. To improve the feasibility and accessibility, there is a need to establish sharable resources among researchers. In related piroplasmid parasites, sporozoite-based inoculums are routinely produced from tick salivary glands, cryopreserved and distributed to other investigators and facilities. For these parasites, sporozoites have been the basis for vaccine development and in vitro cultivation, both of which remain lacking for C. felis research. If infectious sporozoites can be similarly isolated for C. felis, it would significantly broaden our capabilities to study this parasite. Aims of this study was to determine if C. felis sporozoites inoculums collected from the salivary glands of Amblyomma americanum ticks were capable of inducing cytauxzoonosis in naïve cats.Materials and methodsA. americanum nymphs were acquisition-fed on a donor cat chronically infected with C. felis and allowed to molt to adults. Four groups of adult ticks (n = 50/group) were either stimulation-fed for 4 days on naïve cats or were heated at 37 °C for 4 days. After these treatments, salivary glands (SG) of each group of ticks were collected to create inoculums. Infectivity of these inoculums was then tested by subcutaneous injection into naïve cats.ResultsThe two naïve cats used for stimulation feeding and as controls both developed cytauxzoonosis, indicating these groups of ticks were capable of producing infectious sporozoites. Of the 2 cats that were injected with SGs from the stimulation-fed ticks, one cat developed cytauxzoonosis and C. felis infection was confirmed by both light microscopy and PCR. The other cat did not develop cytauxzoonosis and only had equivocal evidence of infection. Neither cat injected with SGs from the heated ticks developed cytauxzoonosis. One of these cats had equivocal evidence of infection and one had no evidence of infection.ConclusionThis study validates the feasibility of collecting infectious sporozoites from C. felis-infected ticks that can be used to infect naïve cats. While this model requires further optimization, it has the potential to expand resources to study C. felis and further advance research in this field.     

Vector competence ofRhipicephalus sanguineus andDermacentor variabilis for American isolates ofBabesia gibsoni

To determine the identity of the tick vector of enzooticBabesia gibsoni in California, two common ixodid ticks were allowed to engorge uponB. gibsoni infected dogs. Sporozoites were observed in the salivary glands of prefed nymphalRhipicephalus sanguineus ticks that fed as larvae onB. gibsoni-infected dogs. A higher proportion (31%) of nymphal ticks that prefed on an uninfected dog for 48 hours contained sporozoites in their salivary glands than did ticks which had fed for 24 hours (13%). Sporozoites were not observed in the salivary glands of prefedR. sanguineus nymphs which were derived from the eggs of adult females that fed on an infected dog, in adults that were fed as nymph on an infected dog, or in the nymphal and adult uninfected controls.Dermacentor variabilis ticks appeared not to become infected. Although attempts to transmitB. gibsoni to susceptible, splenectomized dogs were unsuccessful,R. sanguineus would appear to be the most likely tick vector to maintain this piroplasm in California. This study was supported by grants from the Companion Animal Disease Laboratory, School of Veterinary Medicine, University of California, Davis.     

Genes transcribed in the salivary glands of female Rhipicephalus appendiculatus ticks infected with Theileria parva

We describe the generation of an auto-annotated index of genes that are expressed in the salivary glands of four-day fed female adult Rhipicephalus appendiculatus ticks. A total of 9162 EST sequences were derived from an uninfected tick cDNA library and 9844 ESTs were from a cDNA library from ticks infected with Theileria parva, which develop in type III salivary gland acini. There were no major differences between abundantly expressed ESTs from the two cDNA libraries, although there was evidence for an up-regulation in the expression of some glycine-rich proteins in infected salivary glands. Gene ontology terms were also assigned to sequences in the index and those with potential enzyme function were linked to the Kyoto encyclopedia of genes and genomes database, allowing reconstruction of metabolic pathways. Several genes code for previously characterized tick proteins such as receptors for myokinin or ecdysteroid and an immunosuppressive protein. cDNAs coding for homologs of heme-lipoproteins which are major components of tick hemolymph were identified by searching the database with published N-terminal peptide sequence data derived from biochemically purified Boophilus microplus proteins. The EST data will be a useful resource for construction of microarrays to probe vector biology, vector-host and vector-pathogen interactions and to underpin gene identification via proteomics approaches.     

Effects of Ricinus communis oil esters on salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

This study showed the interference of esters extracted from Ricinus communis in the secretory cycle of salivary glands of Rhipicephalus sanguineus ticks, which consequently caused collateral effects on their feeding process. Ticks attached on hosts which were fed with commercial feed containing different concentrations of R. communis oil esters suffered damages such as cytoplasmic changes in their salivary glands, notably in the acinar cells, impairing the functioning of the acini and accelerating the organs degeneration as a whole. It was found that esters interfered with the activity of cellular secretion by changing the glycoprotein of salivar composition especially in acini II cells. It was also shown that the damages caused by esters in the salivary glands cells of these ectoparasites increased in higher concentrations of the product and degenerative glandular changes were more pronounced.     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Prevalence of Borrelia burgdorferi and Babesia microti in mice on islands inhabited by white-tailed deer.

Borrelia burgdorferi and Babesia microti were isolated from 35 of 51 white-footed mice (Peromyscus leucopus) and meadow voles (Microtus pennsylvanicus) captured on two Narragansett Bay, R.I., islands inhabited by deer, the principal host for the adult stages of the vector tick, Ixodes dammini. Immature ticks parasitized mice from both islands. From 105 mice captured on four other islands not inhabited by deer neither pathogen was isolated, nor were I. dammini found.     

The New Zealand white rabbit: an experimental host for infecting ticks with Lyme disease spirochetes

Efficiency of the New Zealand white rabbit as a host for infecting larval Ixodes dammini, I. pacificus, and I. ricinus with Lyme disease spirochetes was evaluated. Rabbits inoculated with infected midgut suspensions of I. dammini from Shelter Island, New York, or fed upon by infected ticks from the same area, responded with spirochetemias of sufficient concentrations to infect as many as 30 percent of the ticks. When infected ticks were used as indicators, it appeared that spirochetemias persisting for up to ten days occurred as early as the tenth day after inoculation or feeding of ticks.     

Prevalence of Borrelia burgdorferi and Babesia microti in mice on islands inhabited by white-tailed deer

Borrelia burgdorferi and Babesia microti were isolated from 35 of 51 white-footed mice (Peromyscus leucopus) and meadow voles (Microtus pennsylvanicus) captured on two Narragansett Bay, R.I., islands inhabited by deer, the principal host for the adult stages of the vector tick, Ixodes dammini. Immature ticks parasitized mice from both islands. From 105 mice captured on four other islands not inhabited by deer neither pathogen was isolated, nor were I. dammini found.