Molecular screening and genetic diversity of tick-borne pathogens associated with dogs and livestock ticks in Egypt.

BACKGROUND: The Middle East and North Africa (MENA) offer optimal climatic conditions for tick reproduction and dispersal. Research on tick-borne pathogens in this region is scarce. Despite recent advances in the characterization and taxonomic explanation of various tick-borne illnesses affecting animals in Egypt, no comprehensive examination of TBP (tick-borne pathogen) statuses has been performed. Therefore, the present study aims to detect the prevalence of pathogens harbored by ticks in Egypt. METHODOLOGY/PRINCIPAL FINDINGS: A four-year PCR-based study was conducted to detect a wide range of tick-borne pathogens (TBPs) harbored by three economically important tick species in Egypt. Approximately 86.7% (902/1,040) of the investigated Hyalomma dromedarii ticks from camels were found positive with Candidatus Anaplasma camelii (18.8%), Ehrlichia ruminantium (16.5%), Rickettsia africae (12.6%), Theileria annulata (11.9%), Mycoplasma arginini (9.9%), Borrelia burgdorferi (7.7%), Spiroplasma-like endosymbiont (4.0%), Hepatozoon canis (2.4%), Coxiella burnetii (1.6%) and Leishmania infantum (1.3%). Double co-infections were recorded in 3.0% (27/902) of Hy. dromedarii ticks, triple co-infections (simultaneous infection of the tick by three pathogen species) were found in 9.6% (87/902) of Hy. dromedarii ticks, whereas multiple co-infections (simultaneous infection of the tick by ≥ four pathogen species) comprised 12% (108/902). Out of 1,435 investigated Rhipicephalus rutilus ticks collected from dogs and sheep, 816 (56.9%) ticks harbored Babesia canis vogeli (17.1%), Rickettsia conorii (16.2%), Ehrlichia canis (15.4%), H. canis (13.6%), Bo. burgdorferi (9.7%), L. infantum (8.4%), C. burnetii (7.3%) and Trypanosoma evansi (6.6%) in dogs, and 242 (16.9%) ticks harbored Theileria lestoquardi (21.6%), Theileria ovis (20.0%) and Eh. ruminantium (0.3%) in sheep. Double, triple, and multiple co-infections represented 11% (90/816), 7.6% (62/816), and 10.3% (84/816), respectively in Rh. rutilus from dogs, whereas double and triple co-infections represented 30.2% (73/242) and 2.1% (5/242), respectively in Rh. rutilus from sheep. Approximately 92.5% (1,355/1,465) of Rhipicephalus annulatus ticks of cattle carried a burden of Anaplasma marginale (21.3%), Babesia bigemina (18.2%), Babesia bovis (14.0%), Borrelia theleri (12.8%), R. africae (12.4%), Th. annulata (8.7%), Bo. burgdorferi (2.7%), and Eh. ruminantium (2.5%). Double, triple, and multiple co-infections represented 1.8% (25/1,355), 11.5% (156/1,355), and 12.9% (175/1,355), respectively. The detected pathogens' sequences had 98.76-100% similarity to the available database with genetic divergence ranged between 0.0001 to 0.0009% to closest sequences from other African, Asian, and European countries. Phylogenetic analysis revealed close similarities between the detected pathogens and other isolates mostly from African and Asian countries. CONCLUSIONS/SIGNIFICANCE: Continuous PCR-detection of pathogens transmitted by ticks is necessary to overcome the consequences of these infection to the hosts. More restrictions should be applied from the Egyptian authorities on animal importations to limit the emergence and re-emergence of tick-borne pathogens in the country. This is the first in-depth investigation of TBPs in Egypt.

First identification of Cytauxzoon manul in Eurasian lynx (Lynx lynx) in northwestern China.

BACKGROUND: Multiple species of the genera Cytauxzoon and Hepatozoon can infect wild felines, but the diversity of these and other apicomplexan parasites in Eurasian lynx is scarcely known. The aim of this study was to detect Cytauxzoon and Hepatozoon species with molecular methods in Eurasian lynxes and their ticks in northwestern China. METHODS: DNA was extracted from the heart, liver, spleen, lung, and kidney samples of three Eurasian lynxes as well as from their five ixodid ticks. These DNA samples were screened with polymerase chain reactions (PCRs) for Cytauxzoon with the partial cytochrome b gene (CytB), cytochrome c oxidase subunit I gene (COI), and small subunit ribosomal RNA gene (18S rRNA), and Hepatozoon with three different fragments of small subunit ribosomal RNA gene (18S rRNA). PCR products were sequenced, aligned, and phylogenetically analyzed. RESULTS: One adult female of Eurasian lynx (#1, adult female) was co-infected with Cytauxzoon manul and Hepatozoon felis genotype I, while an adult male lynx (#2) was infected with C. manul. Interestingly, H. felis genotype I was both detected in a male cub (#3) and two out of five infesting Hyalomma asiaticum ticks. CONCLUSIONS: For the first time, Cytauxzoon manul is reported here from Eurasian lynx. In addition, H. felis has not been known to occur in this host species in China and Central Asia. Thus, the findings of this study extend our knowledge on the geographical distribution and host range of these haemoprotozoan parasites. Moreover, this is also the first evidence of C. manul and H. felis co-infection in Eurasian lynx.

Pathogens associated with ticks (Acari: Ixodidae) and mammals in the Orinoquia region of Colombia: An approach to understanding vector-pathogen-host interactions.

The hard tick clade (Ixodidae) currently comprises 762 species worldwide (266 Prostriata and 496 Metastriata). A quarter of hard ticks are found in the Neotropical region, and 42 species have been documented in Colombia. Ixodidae species are important vectors of pathogens such as bacteria, helminths, protozoa, and viruses. In tick-borne diseases, vertebrate hosts perform an important role in the transmission, maintenance, and spread of pathogens. Colombia ranks sixth among countries with the highest mammal biodiversity, with a total of 548 species, where some of these species may be involved in pathogen transmission cycles with ticks as vectors. This research evaluated the presence of two genera of bacteria (Borrelia and Rickettsia) and the protozoan (Babesia) in ticks and mammals in the Orinoquia region of Colombia, establishing interaction networks. The information comes from 734 mammals (655 wild and 79 domestic), belonging to 59 species. Tick infestation (n = 1,805) was found with 14.85 % (n = 109) of the examined mammals and corresponds to nine tick species confirmed morphologically and molecularly. To detect pathogens 272 ticks were collected while feeding on 96 mammals; samples from 93 mammals were analyzed. The presence of borreliae from the relapsing fever group (RFG) and the Lyme disease group (LDG) were detected. Rickettsia spp. was detected in ticks and mammals, while Babesia bigemina was only detected in ticks. This research is the first to address the prevalence of zoonotic pathogens in domestic and wild mammals infested with hard ticks in the Department of Arauca, Colombia. Considering that reporting cases of infections with Babesia, Borrelia, and Rickettsia in Colombia is not mandatory, their impact on public health cannot be estimated. This highlights the importance of continuously detecting, confirming, and identifying these and other important pathogens within the "One Health" framework, as they have a significant economic and medical-veterinary impact globally.

Discovery of Colpodella spp. in ticks (Hyalomma dromedarii) infesting camels in southern Egypt.

In Egypt, tick-borne diseases pose a significant threat to human and animal health, and the threat to dromedaries (Camelus dromedarius), the country's dominant camelid species, is of particular concern. These animals are frequently infested with ticks, and may thus develop tick-borne diseases or become reservoirs of tick-borne pathogens. However, there is a paucity of data on tick infestation in Egyptian camels, especially in the south of the country. Accordingly, we aimed to determine the prevalence of tick infestation in southern Egyptian camel populations (in Luxor and Aswan governorates), and identify the hemoprotozoan parasites carried by camel-infesting ticks. Camels were checked for ticks during veterinary examination at quarantine and household checks, and ticks were collected from infested camels for species identification using morphological examination and PCR analyses. Tick and hemoprotozoan species were identified using Basic Local Alignment Search Tool analysis with subsequent confirmation in phylogenetic analyses. All camel-infesting ticks belonged to the species Hyalomma dromedarii, and were clustered with ticks of this species previously found in Egypt in a phylogenetic tree based on the 16S rRNA gene. Molecular analysis targeting the 18S rRNA gene revealed the presence of hitherto undetected hemoprotozoan parasites, Colpodella spp., in 30/297 (10.1 %) camel-infesting ticks. In phylogenetic analysis, these Colpodella spp. were highly homologous (94-98.6 %) with Colpodella spp. previously deposited in GenBank with accession numbers OQ540590Q, MH208621, and GQ411073, which relate to Colpodella spp. previously detected from Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and humans in China. PCR analyses with spherical body protein-4 (SBP-4) gene-specific primers revealed Babesia bovis in 16/297 (5 %) of camel-infesting ticks, however, Babesia bigemina and Theileria annulata were not detected. Here, we report the first detection of Colpodella spp. in H. dromedarii in Egypt. Further epidemiological studies are needed to assess the risk to camels and humans, and the transmission dynamics. Based on the high tick infestation rates in Egyptian camels and the identification of previously unreported protozoan hemoparasites in ticks, we consider that the dromedary should be subject to surveillance as a sentinel species for tick-borne diseases in Egypt. Our findings underline the need for surveillance and collecting data on lesser known pathogens circulating in camel-infesting ticks, as part of a public health strategy for dealing with tick-borne diseases in Egypt.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Redescription of adults and first description of the larva of Haemaphysalis (Rhipistoma) adleri Feldman-Muhsam, 1951 (Acari: Ixodidae), parasite of carnivorans in Western Asia.

The adults of Haemaphysalis (Rhipistoma) adleri Feldman-Muhsam, 1951 (Acari: Ixodidae) are redescribed and the larva of this species is described for the first time here. The adults of H. adleri that we studied were collected from various canid, felid and hyaenid carnivorans (Carnivora: Canidae, Felidae, Hyaenidae) as well as a hedgehog (Erinaceomorpha: Erinaceidae) in Iraq, Israel and West Bank. The males, females and larvae of H. adleri can be differentiated from Haemaphysalis (Rhipistoma) species occurring in the Palearctic portion of West Asia and Egypt as well those in the H. asiatica subgroup by the length of idiosomal setae, development and size of spurs on palpi, dental formula on the hypostome and size of spur on coxae. A lectotype of H. adleri has been designated and the geographic distribution and hosts of this tick species are discussed.

Potential novel Colpodella spp. (phylum Apicomplexa) and high prevalence of Colpodella spp. in goat-attached Haemaphysalis longicornis ticks in Shandong province, China.

Tick-borne Apicomplexan parasites pose a significant threat to both public health and animal husbandry. Identifying potential pathogenic parasites and gathering their epidemiological data are essential for prospectively preventing and controlling infections. In the present study, genomic DNA of ticks collected from two goat flocks (Goatflock1 and Goatflock2) and one dog group (Doggroup) were extracted and the 18S rRNA gene of Babesia/Theileria/Colpodella spp. was amplified by PCR and sequenced. Phylogenetic analysis was conducted based on the obtained sequences. The differences in pathogen positive rates between ticks of different groups were statistically analyzed using the Chi-square or continuity-adjusted Chi-square test. As a result, two pathogenic Theileria (T.) luwenshuni genotypes, one novel pathogenic Colpodella sp. HLJ genotype, and two potential novel Colpodella spp. (referred to as Colpodella sp. struthionis and Colpodella sp. yiyuansis in this study) were identified in the Haemaphysalis (H.) longicornis ticks. Ticks of Goatflock2 had a significantly higher positive rate of Colpodella spp. than those from Goatflock1 (χ2=92.10; P = 8.2 × 10-22) and Doggroup (χ2=42.34; P = 7.7 × 10-11), and a significantly higher positive rate of T. luwenshuni than Doggroup (χ2=5.38; P = 0.02). However, the positive rates of T. luwenshuni between Goatflock1 and Goatflock2 were not significantly different (χ2=2.02; P = 0.16), and so as the positive rates of both pathogens between Goatflock1 and Doggroup groups (P > 0.05). For either Colpodella spp. or T. luwenshuni, no significant difference was found in prevalence between male and female ticks. These findings underscore the potential importance of Colpodella spp. in domestic animal-attached ticks, as our study revealed two novel Colpodella spp. and identified Colpodella spp. in H. longicornis for the first time. The study also sheds light on goats' potential roles in the transmission of Colpodella spp. to ticks and provides crucial epidemiological data of pathogenic Theileria and Colpodella. These data may help physicians, veterinarians, and public health officers prepare suitable detection and treatment methods and develop prevention and control strategies.

Successful treatment with doxycycline monotherapy for human infection with Babesia venatorum (Babesiidae, Sporozoa) in China: a case report and proposal for a clinical regimen.

BACKGROUND: Human babesiosis is a worldwide disease caused by intraerythrocytic protozoa of the genus Babesia. It is transmitted by bites from ixodid ticks, and mechanically transmitted by blood transfusion. It is primarily treated with quinine and/or atovaquone, which are not readily available in China. In this study, we developed a novel treatment regimen involving doxycycline monotherapy in a patient with severe Babesia venatorum infection as an alternative therapeutic medication. The aim of our study is to provide a guidance for clinical practice treatment of human babesiosis. CASE PRESENTATION: A 73-year-old man who had undergone splenectomy and blood transfusion 8 years prior, presented with an unexplained fever, headache, and thrombocytopenia, and was admitted to the Fifth Medical Center of the PLA General Hospital. He was diagnosed with B. venatorum infection by morphological review of thin peripheral blood smears, which was confirmed by multi-gene polymerase chain reaction (PCR), and sequencing of the entire 18s rRNA and partial ß-tubulin encoding genes, as well as isolation by animal inoculation. The doxycycline monotherapy regimen (peros, 0.1 g bisindie) was administered following pharmacological guidance and an effective outcome was observed. The patient recovered rapidly following the doxycycline monotherapy. The protozoan load in peripheral blood samples decreased by 88% in hematocrit counts after 8 days, and negative PCR results were obtained after 90 days of follow-up at the hospital. The treatment lasted for 3 months without any side effects or sequelae. The nine-month follow-up survey of the patient did not reveal any signs of recrudescence or anti-babesial tolerance. CONCLUSIONS: We have reported a clinical case of successful doxycycline monotherapy for human babesiosis caused by B. venatorum, which provides an optional medical intervention for human babesiosis.

HARD TICKS (ACARI: IXODIDAE) INFESTING ARABIAN CAMELS (CAMELUS DROMEDARIUS) IN MEDINA AND QASSIM, SAUDI ARABIA.

Ixodid ticks are hematophagous obligatory ectoparasites that occur worldwide and transmit pathogens to humans and other vertebrates, causing economic livestock losses. The Arabian camel (Camelus dromedarius Linnaeus, 1758) is an important livestock animal in Saudi Arabia that is vulnerable to parasitism by ticks. The diversity and intensity of ticks on Arabian camels in certain localities in the Medina and Qassim regions of Saudi Arabia were determined. One hundred forty camels were examined for ticks, and 106 were infested (98 females, 8 males). A total of 452 ixodid ticks (267 males, 185 females) were collected from the infested Arabian camels. The tick infestation prevalence was 83.1% and 36.4% in female and male camels, respectively (female camels harbored significantly more ticks than did male camels). The recorded tick species were Hyalomma dromedarii Koch, 1844 (84.5%); Hyalomma truncatum Koch, 1844 (11.1%); Hyalomma impeltatum Schulze and Schlottke, 1929 (4.2%); and Hyalomma scupense Schulze, 1919 (0.22%). Hyalomma dromedarii was the predominant tick species in most regions, with a mean intensity of 2.15 ± 0.29 ticks/camel (2.5 ± 0.53 male ticks/camel, 1.8 ± 0.21 female ticks/camel). The proportion of male ticks was higher than that of female ticks (59.1 vs. 40.9%). To the best of our knowledge, this is the first survey of ixodid ticks on Arabian camels in Medina and Qassim, Saudi Arabia.

Ticks and tick-borne microbes identified through passive and active surveillance in Alaska.

Rapid environmental change in Alaska and other regions of the Arctic and sub-Arctic has raised concerns about increasing human exposure to ticks and the pathogens they carry. We tested a sample of ticks collected through a combination of passive and active surveillance from humans, domestic animals, and wildlife hosts in Alaska for a panel of the most common tick-borne pathogens in the contiguous United States to characterize the diversity of microbes present in this region. We tested 189 pooled tick samples collected in 2019-2020 for Borrelia spp., Anaplasma spp., Ehrlichia spp., and Babesia spp. using a multiplex PCR amplicon sequencing assay. We found established populations of Ixodes angustus Neumann (Acari: Ixodidae), Ixodes uriae White (Acari: Ixodidae), and Haemaphysalis leporispalustris Packard (Acari: Ixodidae) in Alaska, with I. angustus found on a variety of hosts including domestic companion animals (dogs and cats), small wild mammals, and humans. Ixodes angustus were active from April through October with peaks in adult and nymphal activity observed in summer months (mainly July). Although no known human pathogens were detected, Babesia microti-like parasites and candidatus Ehrlichia khabarensis were identified in ticks and small mammals. The only human pathogen detected (B. burgdorferi s.s.) was found in a tick associated with a dog that had recently traveled to New York, where Lyme disease is endemic. This study highlights the value of a combined passive and active tick surveillance system to detect introduced tick species and pathogens and to assess which tick species and microbes are locally established.

Transmission of Cytauxzoon felis by injection of Amblyomma americanum salivary glands.

BACKGROUND: Cytauxzoonosis 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 METHODS: A. 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. RESULTS: The 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. CONCLUSION: This 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.

Identification of three members of the multidomain adhesion CCp family in Babesia gibsoni.

Babesia gibsoni is an intraerythrocytic apicomplexan parasite transmitted by Haemaphysalis longicornis and causes canine babesiosis. Within the tick, the Babesia parasite undergoes sexual conjugation and the sporogony process of its life cycle. To control B. gibsoni infection, prompt and effective treatment of acute infections and curing chronic carriers are urgently needed. Gene disruption of Plasmodium CCps resulted in blocking the transition of sporozoites from the mosquito midgut to the salivary glands, showing that these proteins are potential targets for the development of a transmission-blocking vaccine. In this study, we described the identification and characterization of three members of the CCp family in B. gibsoni, named CCp1, CCp2, and CCp3. The B. gibsoni sexual stages were induced in vitro by exposing parasites to xanthurenic acid (XA), dithiothreitol (DTT), and tris (2-carboxyethyl) phosphine (TCEP) at serial concentrations. Among them, 100 µM XA-exposed and cultured at 27 °C without CO2B. gibsoni presented diverse morphologies, including parasites with long projections, gradually increased free merozoites, and aggregated and round forms, indicative of sexual stage induction. Then, the expression of CCp proteins of induced parasites was confirmed by real-time reverse transcription PCR, immunofluorescence, and western blot. The results showed that BgCCp genes were highly significantly increased at 24 h post-sexual stage induction (p < 0.01). The induced parasites were recognized by anti-CCp mouse antisera and anti-CCp 1, 2, and 3 antibodies weakly reacted with sexual stage proteins of expected molecular weights of 179.4, 169.8, and 140.0 KDa, respectively. Our observations on morphological changes and confirmation of sexual stage protein expression will advance elemental biological research and lay the foundation for the development of transmission-blocking vaccines against canine babesiosis.

Diversity of Hepatozoon species in wild mammals and ticks in Europe.

BACKGROUND: Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe. METHODS: Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene. RESULTS: Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22). CONCLUSIONS: Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.

Vitellogenin-2 Accumulation in the Fat Body and Hemolymph of Babesia-Infected Haemaphysalis longicornis Ticks.

The protozoan parasite Babesia spp. invades into tick oocytes and remains in the offspring. The transovarial transmission phenomenon of Babesia in ticks has been demonstrated experimentally, but the molecular mechanisms remain unclear. Babesia invasion into oocytes occurs along with the progression of oogenesis. In the present study, to find the key tick factor(s) for Babesia transmission, we focused on molecules involved in yolk protein precursor (vitellogenin, Vg) synthesis and Vg uptake, which are crucial events in tick oogenesis. With a Haemaphysalis longicornis tick-Babesia ovata experimental model, the expression profiles of Akt, target of rapamycin, S6K, GATA, and Vg, Vg synthesis-related genes, and Vg receptor (VgR) and autophagy-related gene 6 (ATG6), Vg uptake-related genes, were analyzed using real-time PCR using tissues collected during the preovipositional period in Babesia-infected ticks. The expression levels of H. longicornis Vg-2 (HlVg-2) and HlVg-3 decreased in the fat body of Babesia-infected ticks 1 day after engorgement. In the ovary, HlVg-2 mRNA expression was significantly higher in Babesia-infected ticks than in uninfected ticks 1 and 2 days after engorgement and decreased 3 days after engorgement. HlVgR expression was significantly lower in Babesia-infected ticks than in uninfected ticks 2 and 4 days after engorgement. HlATG6 had a lower gene expression in Babesia-infected ticks compared to uninfected ticks 2 days after engorgement. Additionally, western blot analysis using protein extracts from each collected tissue revealed that H. longicornis Vg-2 (HlVg-2) accumulate in the fat body and hemolymph of Babesia-infected ticks. These results suggest that Vg uptake from the hemolymph to the ovary was suppressed in the presence of B. ovata. Moreover, HlVg-2 knockdown ticks had a lower detection rate of B. ovata DNA in the ovary and a significant reduction of B. ovata DNA in the hemolymph compared with control ticks. Taken together, our results suggest that accumulated HlVg-2 is associated with Babesia infection or transmission in the tick body. These findings, besides previous reports on VgR, provide important information to elucidate the transovarial transmission mechanisms of pathogens in tick vectors.

Morphological identification and molecular characterization of economically important ticks (Acari: Ixodidae) from North and North-Western Egypt.

Ticks represent a major source of growing economic and public health concern, especially in the tropics and sub-tropics. Towards evidencing ticks' invasion in the North and North-Western parts of Egypt, the present study aimed to investigate the morpho-molecular aspects of those ectoparasites using stereomicroscopy, scanning electron microscopy (SEM) and sequencing of the mitochondrial 16S ribosomal DNA gene (rDNA) and nuclear second internal transcribed spacer (ITS2). Our analysis confirmed the presence and well-distribution of Rhipicephalus sanguineus s.l. infesting dogs and sheep (Alexandria governorate), Rhipicephalus annulatus infesting cattle (Beheira governorate), and Hyalomma dromedarii infesting camels (Marsa Matruh governorate) from North/North-Western Egypt. 16S rDNA and ITS2 sequences of the ticks were amplified using universal and gene-specific sets of primers, sequenced and analyzed. Lengths of amplified 16S rDNA sequences in all examined tick species were found to be similar in size (approximately 460 bp); however, they differed in base pair constitutions, whereas ITS2 lengths were 1,500 bp, 1,550 bp, and 1,800 bp for Rh. annulatus, Rh. sanguineus s.l., and Hy. dromedarii, respectively. Phylogenetically, based on the 16S rDNA results, Rh. sanguineus s.l. ticks clustered with the southeastern Europe lineage from Romania and Greece, Rh. annulatus ticks were similar to Turkish populations, and Hy. dromedarii were close to the isolates from Tunisia. Similarly, based on ITS2 sequences, Rh. sanguineus s.l. from dogs were showing 99% similarity to Nigerian populations; however, those collected from sheep were closer to Iranian populations with 4.1% nucleotide divergence between the two populations of different hosts. Rh. annulatus ticks were identical to a population from Romania, whereas Hy. dromedarii was close by 99.7% similarity to a population from Kenya. This is the first study reporting nucleotide sequences of 16S rDNA and ITS2 in integration with morphological identification of ticks from this part of Egypt.

Discordant phylogeographic patterns between the tortoise tick Hyalomma aegyptium and their Testudo graeca hosts.

Tortoise ticks, Hyalomma aegyptium, are considered so strongly associated with their hosts that they are even used as indirect indicators for them. In such a case, a robust pattern of congruence between host and parasite could be expected, with phylogeographic breaks within the host being reflected in their parasites. We sequenced two mitochondrial partial gene regions (12S rRNA and Cytochrome Oxidase 1) from ticks across northern Africa and Anatolia, and compared patterns of variation with those identified in its main host, Testudo graeca. Two distinct haplogroups were identified, both of which were found distributed across much of northern Africa. This pattern does not reflect the known variation within the host, which has multiple, geographically disjunct subspecies in this region, but rather the major climatic zones. This relationship can be explained by adaptive processes to environmental conditions influenced by the climate, as well as by the spatial structure of the communities of tick potential hosts in larval and nymphal stages. Extensive anthropogenic movement of tortoises may also obscure congruence patterns between H. aegyptium ticks and their hosts.

Raman-based identification of tick species (Ixodidae) by spectroscopic analysis of their feces.

Ticks are blood-feeding parasites that vector a large number of pathogens of medical and veterinary importance. There are strong connections between tick and pathogen species. Timely detection of certain tick species on cattle can cease the spread of numerous devastating diseases such as Bovine babiesiosis and anaplasmosis. Detection of ticks is currently performed by slow and laborious scout-based inspection of cattle. In this study, we investigated the possibility of identification of tick species (Ixodidae) based on spectroscopic signatures of their feces. We collected Raman spectra from individual grains of feces of seven different species of ticks. Our results show that Raman spectroscopy (RS) allows for highly accurate (above 90%) differentiation between tick species. Furthermore, RS can be used to predict the tick developmental stage and differentiate between nymphs, meta-nymphs and adult ticks. We have also demonstrated that diagnostics of tick species present on cattle can be achieved using a hand-held Raman spectrometer. These findings show that RS can be used for non-invasive, non-destructive and confirmatory on-site analysis of tick species present on cattle.

Nested PCR assay for detection of Theileria annulata in Hyalomma anatolicum infesting cattle from coastal Odisha, India.

Ticks are economically important obligatory blood feeding arthropods that have a pivotal role in transmission of infection. The present study was conducted in ixodid ticks collected from four districts of coastal Odisha, India to investigate the prevalence of Theileria annulata. Adult semi engorged Hyalomma anatolicum ticks (n = 178) were dissected, the salivary gland was isolated and DNA was extracted. A nested PCR targeting the Tams1 gene of T. annulata, utilizing two sets of primers (N516F, N517R, and Ta14136iF, Ta249R) was utilized for detection of the parasite. The PCR products were then sequenced and subjected to BLAST analysis, alignment, and phylogenetic study. Two sequences deposited in GenBank were assigned Accession No MH477290.1 and Accession No MH477291.1. The molecular investigation of T. annulata revealed an overall prevalence of 14.6% in tick vectors, and nested PCR was found to have significant (p < 0.05) higher results than primary PCR. A significant higher presence (p < 0.05) was recorded in female ticks compared with male ticks. This is the first report of detection of the parasite in tick vectors in the state of Odisha.

Direct injection of Amblyomma americanum ticks with Cytauxzoon felis.

Cytauxzoon felis is a tick-borne hemoprotozoan parasite that causes life-threatening disease in domestic cats in the United States. Currently, the platforms for C. felis research are limited to natural or experimental infection of domestic cats. This study aims to develop an alternative model by infecting Amblyomma americanum ticks with C. felis via direct injection. Amblyomma americanum adults were injected with C. felis-infected feline erythrocytes through two routes: directly into the digestive tract through the anal pore (IA injection), or percutaneously into the tick hemocoel (IH injection). RNAscope® in situ hybridization (ISH) was used to visualize the parasites within the ticks at different time points after injection. Four months after injection, ticks were divided into 3 infestation groups based on injection methods and inoculum type and fed on 3 naïve cats to assess the ticks' ability to transmit C. felis. Prior to the transmission challenge, selected ticks from each infestation group were tested for C. felis RNA via reverse transcription-PCR (RT-PCR). In both IA- and IH-injected ticks, ISH signals were observed in ticks up to 3 weeks after injection. The number of hybridization signals notably decreased over time, and no signals were detected by 4 months after injection. Prior to the transmission challenge, 37-57% of the sampled ticks were positive for C. felis RNA via RT-PCR. While the majority of injected ticks successfully attached and fed to repletion on all 3 cats during the transmission challenge, none of the cats became infected with C. felis. These results suggest that injected C. felis remained alive in ticks but was unable to progress to infective sporozoites after injection. It is unclear why this infection technique had been successful for other closely related tick-borne hemoprotozoa and not for C. felis. This outcome may be associated with uncharacterized differences in the C. felis life cycle, the lack of the feeding or molting in our model or absence of gametocytes in the inoculum. Nonetheless, our study demonstrated the potential of using ticks as an alternative model to study C. felis. Future improvement of a tick model for C. felis should consider other tick species for the injection model or utilize infection methods that more closely emulate the natural infection process.

Surveillance and Molecular Identification of Borrelia Species in Ticks Collected at U.S. Army Garrison Humphreys, Republic of Korea, 2018-2019.

Tick-borne pathogens are contributing factors for the increased incidence of vector-borne diseases throughout the world, including Lyme borreliosis, one of the most prevalent spirochetes belonging to the Borrelia burgdorferi sensu lato group. The present study focused on the detection of Borrelia species from hard ticks collected at U.S. Army Garrison Humphreys, Republic of Korea (ROK), using molecular and genotypic analyses. Tick-borne disease surveillance was conducted from January to December, 2018-2019. A total of 24,281 ticks (2 genera and 5 species) were collected from road-killed Korean Water deer (KWD) and by tick drag. Haemaphysalis longicornis (92.0%) was the most commonly collected species, followed by Haemaphysalis flava (4.9%), Ixodes nipponensis (3.1%), Haemaphysalis phasiana (0.07%), and Haemaphysalis japonica (<0.01%). The ospA gene sequences of Borrelia afzelii were detected in 12/529 pools of I. nipponensis. Three and one pools were positive for B. afzelii and Borrelia miyamotoi, respectively, using the 16s rRNA gene. None of the pools of Haemaphysalis ticks collected from KWD or by tick drag were positive for Borrelia species. I. nipponensis was collected throughout the year from KWD and from February to November by tick drag, suggesting that they were active throughout the year, and expanding the risk period for acquiring Lyme borreliosis and Borrelia relapsing fever in the ROK. This study assessed disease risk factors associated with the prevalence of Lyme disease in ticks collected from KWD and by tick drag using molecular analysis. These results provide an understanding and awareness into the prevalence and molecular characteristics of Borrelia species in the ROK.