Prototypes of Hyalomma scupense Schulze, 1918 and H. detritum Schulze, 1919 (Acari: Ixodidae) in connection with microevolution within the genus

Type series of two species of the genus Hyalomma Koch, 1844 deposited in the Museum of Zoology in Berlin have been examined. Examination of the H. detritum Schulze, 1919 holotype has shown that this name is actually a junior synonym (syn. nov.) of H. marginatum turanicum Pomerantsev, 1946. At the same time it was also found out that the paratypes of H. detritum are conspecific to the syntypes of H. scupense Schulze, 1818, among which the lectotype has been designated. Taxonomic errors of Schulze and other authors, which had led to a worldwide use in acorological literature the name H. detritum instead of the valid name H. scupense, are discussed. It is suggested that the reasons of microevolution within the polymorphic species H. scupense could be explained by unequal climatic conditions. Microevolutionary process in this species is most well expressed in a tendency to reduce the number of contacts with a host during the life cycle. It has resulted in the reformation of the two-host cycle into the one-host cycle.     

Electron microscopic studies of the development of kinetes in Theileria annulata Dschunkowsky & Luhs, 1904 (Sporozoa, Piroplasmea).

Gamogony of Theileria annulata Dschunkowsky & Luhs occurs within the intestine of nymphs of the tick Hyalomma anatolicum excavatum Koch. After the 5th day post repletionem (p.r.) of the ticks spherical and ovoid parasites were found within the intestinal cells. These stages were thought to represent fertilized macrogametes. These underwent a transformation process leading ultimately to the differentiation of a motile stage, the kinete, which leaves the intestinal cells on the 14th-17th day p.r. and penetrates the alveoles of the salivary glands. The transformation of the stationary into a motile stage takes place by formation of a growing protrusion (= anlage) into an inner, enlarging vacuole. During this process the limiting membrane of the vacuole serves as the outer membrane of the developing motile stage, whereas the 2 inner membranes of its pellicle are newly formed. The steps of this differentiation in T. annulata are compared to the process of ookinete formation in haemosporina.     

Electron Microscopic Studies of the Development of Kinetes in Theileria annulata Dschunkowsky & Luhs, 1904 (Sporozoa, Piroplasmea)

SYNOPSIS. Gamogony of Theileria annulata Dschunkowsky & Luhs occurs within the intestine of nymphs of the tick Hyalomma anatolicum excavatum Koch. After the 5th day post repletionem (p.r.) of the ticks spherical and ovoid parasites were found within the intestinal cells. These stages were thought to represent fertilized macrogametes. These underwent a transformation process leading ultimately to the differentiation of a motile stage, the kinete , which leaves the intestinal cells on the 14th-17th day p.r. and penetrates the alveoles of the salivary glands. The transformation of the stationary into a motile stage takes place by formation of a growing protrusion (= anlage) into an inner, enlarging vacuole. During this process the limiting membrane of the vacuole serves as the outer membrane of the developing motile stage, whereas the 2 inner membranes of its pellicle are newly formed. The steps of this differentiation in T. annulata are compared to the process of ookinete formation in haemosporina.     

Light microscopic studies on the development of Theileria annulata (Dschunkowsky and Luhs, 1904) in Hyalomma anatolicum excavatum (Koch, 1844). II. The development in haemolymph and salivary glands (author's transl)]

Fully differentiated kinetes, average length 17.6 micrometer, appeared in the haemolymph of engorged nymphs usually 17 to 20 days after repletion. Kinetes were observed at first in the salivary glands on day 18 after repletion. The kinetes then transformed into fission bodies of about 10 micrometer in diameter, mainly in type III alveoli and less frequently in type II alveoli. The fission bodies grew up to a size of about 20 micrometer after several divisions of their nucleus. At this time the ticks moulted and no further development occurred until activation. Shortly before infestation the salivary glands began to proliferate, and rapid growth of the fission bodies was observed, especially in young ticks where development of 'infective particles' ('sporozoites') was concluded within two days. Development in feeding adult ticks apparently occurred in four major steps: (1) Division of primary fission bodies (sporonts) into numerous secondary fission bodies ('primary sporoblasts'), (2) division of secondary fission bodies into tertiary fission bodies ('secondary sporoblasts'), (3) production of particles ('sporozoites') by tertiary fission bodies and release of particles into the saliva, and (4) degeneration of fission bodies and their host cell but further release of particles. The host cell was stimulated to giant growth, thus its diameter increased, on average, from 15 to 110 micrometer. Heavy infections resulting from parasitaemias of greater than 40% caused disease and mortality in the tick population. Development was much retarded by aging. In ticks starved for six months 'sporozoites' did not develop before day five to seven of infestation. 'Sporozoites' did not develop before day five to seven of infestation. 'Sporozoites' may not develop at all in six to nine month old female ticks during the infestation period. The significance of the described developmental stages of T. annulata was discussed and a sexual generation postualted. The hypothetic development of T. annulata in its tick vector was illustrated.     

Prevalence of Theileria in the tick Hyalomma detritum detritum in the Doukkala region,Morocco

Abstract. The overall prevalence of Theileria species, mainly, if not exclusively, T.annulata, in 901 Hyalomma detritum detritum collected from cattle in the Doukkala region of Morocco over a period of 2 years was 21.5%. The quantity of infection (number of sporoblasts per infected tick) followed the negative binomial distribution with between one and 250 sporoblasts per infected tick. Infected ticks were found in eight of fourteen areas examined whilst T.annulata was present in all fourteen. There were significant differences in both the prevalence and the quantity of infection between ticks collected from different farms, and between nymphs collected in the autumn from these farms, and moulted in the laboratory, and adults collected in the following summer. The prevalence, but not the quantity, of infection was higher in female than in male ticks. No correlations were established between infection of engorged nymphs and the breed, sex and Theileria piroplasm parasitaemia of the host animal. However, calves infected a greater proportion of nymphs than adult cattle and the heavier the infestation of nymphs on an animal, up to a plateau, the higher the prevalence of infection in those nymphs. There were no differences in infection between ticks moulted at 24^oC and 37^oC, after the engorged nymphs had been stored at 18^oC to simulate over-wintering.     

Detection of the protozoan parasite Theileria annulata in Hyalomma ticks by the polymerase chain reaction

Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.     

Theileria annulata and Babesia ovis: ultracytochemical lactic dehydrogenase activity of sporozoites in salivary glands of female ticks, Hyalomma anatolicum excavatum and Rhipicephalus bursa

The occurrence of a marker enzyme of glycolysis, lactic dehydrogenase (LDH) (EC 1.1.1.27.), was studied ultracytochemically in sporozoites of Babesia ovis in the tick Rhipicephalus bursa and in sporozoites of Theileria annulata in the tick Hyalomma anatolicum excavatum. Female ticks infected transstadially were fed on rabbits and dissected 3–5 days post infestationem. The salivary glands were removed and incubated in the cytochemical medium unfixed or after fixation in buffered paraformaldehyde solution. A modified ferricyanide medium adjusted to pH 6.5 was used for incubation. Controls were performed by preincubating specimens in 10^?3 M iodine or by omitting NAD⁺ or lactate in the incubation medium. Following incubation, the specimens were fixed in buffered solutions of paraformaldehyde or glutaraldehyde, postfixed in osmium tetroxide, and embedded in Durcupan ACM. Mature “schizonts” consisting of an abundant number of sporozoites were examined in both piroplasmean species. In sporozoites of B. ovis the final enzymic product was deposited within the nucleus. No cytoplasmic reaction was observed. However, the membrane delimiting the schizont from the adjacent glandular cells was distinctly reactive. In T. annulata reactivity was usually confined to the cytoplasm. Sometimes, a reaction within mitochondria could be observed. The reaction product had formed aggregations which often appeared to be attached to micronemes. There was no nuclear reactivity in this species.The results suggest the existence of a glycolytic metabolic pathway with different subcellular localizations in sporozoites of the two piroplasmean species.     

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata

HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata‐infected counterpart was utilized to test the effects of geldanamycin and the derivative 17‐AAG. T. annulata‐infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17‐AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.     

Detection of Theileria annulata by the PCR in ticks (Acari: Ixodidae) collected from cattle in Mauritania

We report on the detection of Theileria annulata in infected Hyalomma ticks by the PCR using primers derived from the gene encoding the 30 kDa major merozoite surface antigen (Tams1–1). No inhibition of the PCR was observed and as little as 0.1 pg of parasite DNA, corresponding to 12 sporozoites, could be detected in non-infected tick DNA samples, spiked with T. annulata genomic DNA. Hyalomma dromedarii ticks, fed on a calf experimentally infected with T. annulata, were used to validate the PCR further. The infection rate in the adult ticks, fed as nymphs during the febrile reaction, was high (62%), dropped to zero for 1 day in tick batches that engorged after treatment with ButalexTM and increased to 30% 2 days later and 38% of the ticks acquired the infection after feeding as nymphs during a carrier state piroplasm parasitaemia of less than 0.1%. As an internal control, 16S tick rDNA sequences could be amplified from T. annulata-negative tick samples. Finally, 202 adult ticks from Mauritania, collected from zebu cattle carrying low levels of Theileria piroplasms, were tested by the PCR. Thirty-eight out of 52 (73%) and 17 out of 30 (57%) H. dromedarii from the Gorgol and Trarza regions, respectively and two out of 30 (7%) Hyalomma marginatum rufipes from the Gorgol region were positive. Hyalomma marginatum rufipes, Rhipicephalus evertsi evertsi and Rhipicephalus guilhoni from the Trarza region were negative. These findings confirm that H. dromedarii is the main vector of T. annulata in Mauritania and that the PCR is a useful method of determining the infection rates in ticks collected from cattle carrying low levels of T. annulata piroplasms.     

Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation‐like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection‐associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta‐miR‐181a and bta‐miR‐181b, we identified putative messenger RNA targets and confirmed the interaction of bta‐miR181a with ICAM‐1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.     

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.     

Biochemical, Hematological, and Electrocardiographic Changes in Buffaloes Naturally Infected with Theileria annulata

Changes in selected blood and serum components and electrocardiography (ECG) were investigated in 20 adults (13 females and 7 males) of water buffaloes suffering from severe theileriosis. The age of all animals used in this study ranged 1.5-5 yr. Theileriosis was diagnosed by observation of parasites in the peripheral blood and the presence of schizonts in lymphocytes that were provided from swollen lymph nodes. Statistically significant decreases were observed in the means of RBC, WBC, and packed cell volume (PCV) in blood of infected animals. The means levels of sodium, calcium, phosphorus, and potassium of infected animals were lower than healthy animals, but only the decrease of potassium was significant. The mean serum activities of aspartate transferase and alanine aminotransfrase were significantly higher than in uninfected animals. Three cases had atrial premature beat, 2 cases had sinus tachycardia, 2 had sinus arrhythmia, and 1 had first degree of atrioventricular block in ECG. The present study showed that T. annulata infection in cattle is associated with hematological and biochemical, and ECG changes.     

Morphology of spiracles in adult Hyalomma truncatum ticks (Acari; Ixodidae)

Scanning electron microscopical investigations of fractures and corrosion casts of spirales in adult ticks of Hyalomma truncatum revealed a three-part structure consisting of the spiracular plate forming the outer part followed by the subostial space, which leads into the atrial chamber from which the main tracheal trunks arise. The spiracular plate sonsists of a thin surface plate perforated by aeropyles, an underlying interpedicellar space formed by pedicels and an inner thick base plate. The surface plate is subdivided into a porous and a non-porous area. The macula is surrounded by the porous area and cleft by the ostium, which is bounded by a lip. The lip rests on a stalk which passes through the subostial space and forms the lateral wall of the atrial chamber. The interpedicellar space is chambered comprising four types of chambers. Large pyriform chambers (type 1) open to the atmosphere via a large aeropyle and are connected at their base with a duct traversing the base plate. They correspond numerically and in their position with the large aeropyles and the ducts of the base plate. Each chamber is surrounded by four to six medium-sized tubular chambers (type 2) which are closed at both ends. Small tubular chambers (type 3) open to the atmosphere via a small aeropyle, are closed at their base and correspond in number and position to the small aeropyles. Elongated chambers (type 4) are arranged in two to three rows around the subostial space and are closed at both ends. The front row communicates with the subostial space via large gaps. All chambers interconnect with each other by slit-like fenestrations. Below the macula and surrounding the stalk is the subostial space. Over the medial half, the subostial space opens into the atrial chamber. The lateral wall of the atrial chamber is thick, whereas the opposite wall is thin, folded and can be everted and inverted. Inverted, the medial wall closes up the opening to the subostial space and the main tracheal trunks. The base of the atrial chamber sonsists of the openings of the main tracheal trunks only. It is concluded that the aeropyles constitute the functional openings of a spiracle, the interpedicellar space and the subostial space act as diffusion barrier and the atrial chamber is exclusively responsible for the motory process of in- and expiration and is the only closing device of the spiracle.     

Ultrastructural demonstration of succinic dehydrogenase and cytochrome oxidase activity in sporozoites of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in salivary glands of tick vectors (Rhipicephalus bursa, Hyalomma anatolicum excavatum)

Salivary gland stages ("sporozoites") of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in female ixodid ticks were studied for ultracytochemical activity of the respiratory enzymes, succinic dehydrogenase (SDH), and cytochrome oxidase. Both SDH and cytochrome oxidase were demonstrated in the sporozoites and the mitochondria in these stages. Identified in this way the final reaction product of SDH was located mainly at the inner side of the mitochondrial boundary, though it was also visible in the internal space of the organelle. Cytochrome oxidase activity always was confined to the wall of mitochondria. This enzyme was demonstrated also in the erythrocyte stage of B. ovis. The cytochemical results indicate respiratory potential of the piroplasmean stages studied. Cristate or typical protozoan mitochondria have not been observed in sporozoites of Babesia or Theileria. This report is the first demonstration of mitochondria, or mitochondrialike activity in Babesia.