A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Molting incidents of Hyalomma spp. carrying human pathogens in Germany under different weather conditions.

BACKGROUND: Hyalomma marginatum and H. rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa to central Asia but may also be found in Central and Northern Europe through introduction by migratory birds. METHODS: Ticks were collected while feeding or crawling on animals and humans, or from the environment, in different regions in Germany, between 2019 and 2021 in a citizen science study and from 2022 to 2023 in the wake of this study. RESULTS: From 2019 to 2023, a total of 212 Hyalomma adult ticks were detected in Germany. This included 132 H. marginatum and 43 H. rufipes ticks sent to research institutions and 37 photographic records that were only identified to genus level. The number of detected ticks varied over the years, with the highest number of 119 specimens recorded in 2019, followed by 57 in 2020. Most of the specimens were collected from horses, while some were collected from other animals, humans or found crawling on human clothes or other objects inside or outside houses. The screening of 175 specimens for Crimean-Congo hemorrhagic fever virus and of 132 specimens for Babesia/Theileria spp. by PCR gave negative results, while human-pathogenic Rickettsia were detected in 44% (77/175) of the total samples. Subsequent amplicon sequencing and phylogenetic analysis of representative samples determined the species of 41 Rickettsia aeschlimannii and one R. slovaca sequences. CONCLUSIONS: Analysis of climatic factors indicated a significantly higher probability of Hyalomma occurrence at locations with higher average spring temperature during the years 2019 and 2020 compared to randomly generated pseudo-absence locations. Dry and hot conditions probably facilitated Hyalomma nymphs' survival and molting into adults during these years.

In vitro feeding of all life stages of two-host Hyalomma excavatum and Hyalomma scupense and three-host Hyalomma dromedarii ticks.

Ticks are blood-sucking ectoparasites and can transmit various pathogens of medical and veterinary relevance. The life cycle of ticks can be completed under laboratory conditions on experimental animals, but the artificial feeding of ticks has attracted increased interest as an alternative method. This study represents the first report on the successful in vitro feeding of all life stages of two-host tick species, Hyalomma scupense and Hyalomma excavatum, and the three-host tick Hyalomma dromedarii. The attachment and engorgement rates of adults were 84% (21/25) and 76% (19/25) for H. scupense females. For adult H. excavatum and H. dromedarii, 70% (21/30) and 34.4% (11/32) of the females attached and all attached females successfully fed to repletion. The oviposition rates of the artificially fed females were 36.4%, 57.1% and 63.1% for H. dromedarii, H. excavatum and H. scupense, respectively, with a reproductive efficiency index varying between 44.3 and 60.7%. For the larvae, the attachment and engorgement rates were 44.2% (313/708) and 42.8% (303/708) for H. dromedarii, 70.5% (129/183) and 56.8% (104/183) for H. excavatum and 92.6% (113/122) and 55.7% (68/122) for H. scupense. The attachment and engorgement rates for the nymphs were 90.2% (129/143) and 47.6% (68/143) for H. dromedarii, 66.7% (34/51) and 41.2% (21/51) for H. excavatum, and 44.1% (30/68) and 36.8% (25/68) for H. scupense. Molting rates of the immature stages varied between 71.3% (216/303) and 100% (68/68) for the larvae and between 61.9% (13/21) and 96% (24/25) for the nymphs. The successful in vitro feeding of all stages of the three Hyalomma species makes this method a valuable tool for tick research, with potential applications in studies on the pathogens transmitted by these tick species such as Theileria annulata.

Molecular survey on vector-borne pathogens in clinically healthy stray cats in Zaragoza (Spain).

BACKGROUND: In Europe, feline vector-borne infections are gaining importance because of the changing climate, expanding habitats of potential vectors and expanding pathogen reservoirs. The main objective of this study was to assess the prevalence of vector-borne pathogens (VBPs) in stray cats in Zaragoza, Spain, and to investigate potential risk factors for infection, including feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). METHODS: Blood samples from stray cats presented to the veterinary faculty in Zaragoza between February 2020 and 2022 were tested by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Ehrlichia canis, Rickettsia spp., haemotropic Mycoplasma spp., Hepatozoon spp., Leishmania infantum, piroplasms and microfilariae at the LABOKLIN laboratory. The cats were also tested for FeLV and FIV by PCR. RESULTS: Nearly half of the cats (158/332, 47.6%) were positive for at least one VBP. Hepatozoon spp. were detected in 25.6%, haemotropic Mycoplasma spp. in 22.9%, B. henselae in 9.3% and L. infantum in 2.1% of the cats. Male sex had a statistically significant association with test results for haemotropic Mycoplasma spp. (odds ratio 1.38 [1.21;1.57]); regionality with Hepatozoon spp., B. henseale and FIV; and seasonality with Hepatozoon spp., haemotropic Mycoplasma spp., L. infantum and FeLV (P ≤ 0.05 each). A strong positive correlation was reported for the amount of rainfall and the number of cats that tested positive for Hepatozoon spp. (ρ = 753, P = 0.05). None of the cats tested positive for A. phagocytophilum, A. platys, E. canis, Rickettsia spp., piroplasms, or microfilariae. Co-infections with multiple VBPs were detected in 56 out of 332 cats (16.9%). Thirty-one of the 332 cats included in the study (9.3%) tested positive for FeLV (6.9%) and for FIV (3.6%). In 20/31 cats (64.5%) that tested positive for FeLV/FIV, coinfections with VBP were detected (P = 0.048, OR 2.15 [0.99; 4.64]). CONCLUSIONS: VBPs were frequently detected in stray cats in Zaragoza. In particular, regionality and seasonality had a statistically significant association with PCR results for most VBPs included in the study.

Participatory survey of risk factors and pathways for Rift Valley fever in pastoral and agropastoral communities of Uganda.

To assess pastoralists' and agropastoralists' knowledge on Rift Valley fever (RVF), participatory epidemiological studies were conducted with 215 livestock keepers and 27 key informants in Napak, Butebo, Isingiro and Lyantonde districts, Uganda, between January and February 2022. Livestock keepers in all four districts had knowledge of RVF and even had local names or descriptions for it. Pastoralists and agropastoralists possessed valuable knowledge of RVF clinical descriptions and epidemiological risk factors such as the presence of infected mosquitoes, living in flood-prone areas, and excessive rainfall. RVF was ranked among the top ten most important cattle diseases. Pastoralists called RVF Lonyang, symbolizing a disease associated with jaundice, high fever, abortions in pregnant cows, and sudden death in calves. Key informants identified infected domestic animals, the presence of infected mosquitoes, livestock movement and trade, and infected wild animals as risk pathways for the introduction of RVF into an area. Drinking raw blood and milk was perceived as the most likely pathway for human exposure to RVF virus; while the highest consequence was high treatment costs. The results indicate that pastoralists provided key epidemiological information that could be essential for designing an effective national RVF surveillance and early warning system.

Ecological and subject-level drivers of interepidemic Rift Valley fever virus exposure in humans and livestock in Northern Kenya.

Nearly a century after the first reports of Rift Valley fever (RVF) were documented in Kenya, questions on the transmission dynamics of the disease remain. Specifically, data on viral maintenance in the quiescent years between epidemics is limited. We implemented a cross-sectional study in northern Kenya to determine the seroprevalence, risk factors, and ecological predictors of RVF in humans and livestock during an interepidemic period. Six hundred seventy-six human and 1,864 livestock samples were screened for anti-RVF Immunoglobulin G (IgG). Out of the 1,864 livestock samples tested for IgG, a subset of 1,103 samples was randomly selected for additional testing to detect the presence of anti-RVFV Immunoglobulin M (IgM). The anti-RVF virus (RVFV) IgG seropositivity in livestock and humans was 21.7% and 28.4%, respectively. RVFV IgM was detected in 0.4% of the livestock samples. Participation in the slaughter of livestock and age were positively associated with RVFV exposure in humans, while age was a significant factor in livestock. We detected significant interaction between rainfall and elevation's influence on livestock seropositivity, while in humans, elevation was negatively associated with RVF virus exposure. The linear increase of human and livestock exposure with age suggests an endemic transmission cycle, further corroborated by the detection of IgM antibodies in livestock.

De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection.

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick's enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum.

Decontamination protocols affect the internal microbiota of ticks.

Studies on the microbiota of ticks have promoted hypotheses about the combined effects of the bacterial community, its functional contributions to the tick's physiology or probable competition effects with some tick-borne pathogens. However, knowledge on the origin of the microbiota of newly hatched larvae is missing. This study aimed to elucidate the source(s) of the microbiota in unfed tick larvae, addressing the composition of the "core microbiota" and the best ways to decontaminate eggs for microbiota studies. We applied laboratory degree bleach washes and/or ultraviolet light treatments on engorged Rhipicephalus australis females and/or their eggs. No significant effects of these treatments on the reproductive parameters of females and the hatching rates of eggs were observed. However, the different treatments did show striking effects on the composition of the microbiota. The results indicated that bleach washes disrupted the internal tick microbiota in females, implying that bleach may have entered the tick and subsequently affected the microbiota. Furthermore, the analyses of results demonstrated that the ovary is a main source of tick microbiota, while the contribution of Gené's organ (a part of the female reproductive system that secretes a protective wax coat onto tick eggs) or the male's spermatophore requires further investigation. Further studies are needed to identify best practice protocols for the decontamination of ticks for microbiota studies.

Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine.

INTRODUCTION: Borrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection. METHOD: Label-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies. RESULTS: We identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host. CONCLUSION: Using quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.

Ticks in the metropolitan area of Berlin, Germany.

A high-resolution city map showing the geographic distribution of 12 tick species (Acari: Argasidae, Ixodidae) that have been recorded from the metropolitan area of Berlin, Germany is presented. A total of 237 tick locations was mapped. These include ten ixodid tick species: Dermacentor reticulatus, Haemaphysalis concinna, Hyalomma rufipes, Ixodes ricinus, Ixodes canisuga, Ixodes hexagonus, Ixodes arboricola, Ixodes frontalis, Ixodes trianguliceps and Rhipicephalus sanguineus sensu lato. The two tick species Hy. rufipes and R. sanguineus s.l. are not endemic to Berlin. Hyalomma rufipes ticks are introduced in Europe with migratory birds from Africa every spring. Rhipicephalus sanguineus s.l. are introduced to Central Europe with dogs that had travelled to or were imported from countries where this tick is endemic. In Germany, they are able to develop and reproduce inside heated buildings. Occurrences of two soft tick species, the pigeon tick Argas reflexus and the short-legged bat tick Carios vespertilionis were also mapped. Other tick species that are likely to be endemic to Berlin and its environs, but for which documented findings or geographical coordinates are lacking, are mentioned. These include the long-legged bat tick I. vespertilionis and the marten tick I. rugicollis documented in Brandenburg, the federal state surrounding Berlin. It can be assumed that if appropriate field studies are carried out, these tick species will also be found in the metropolitan area of Berlin. The high-resolution mapping of all tick species found in a city (like Berlin) forms the basis for further investigations into the impact of climate change and changing land use on ticks and tick-borne diseases, precisely in those habitats where most people will live in the future.

First evidence of vertical Hepatozoon canis transmission in dogs in Europe.

BACKGROUND: Hepatozoon canis is a protozoal agent that is known to be transmitted by oral uptake of H. canis-infected Rhipicephalus sanguineus sensu lato ticks in dogs. Vertical transmission of H. canis has only been described once in a study evaluating dogs from Japan. The aim of this study was to investigate the parasitological status of puppies from a bitch that had tested positive for Hepatozoon spp. prior to giving birth. FINDINGS: A 4-year-old, female, pregnant dog imported from Italy (Sardinia) to Germany showed clinical signs of lethargy and tachypnoea and tested positive for H. canis by PCR. The dog gave birth to eight puppies, one of which was stillborn and another that had to be reanimated. Haematology, buffy coat analysis and a biochemistry profile were performed for each dog. EDTA-blood of the surviving seven puppies and bone marrow, liver, spleen, amniotic fluid, and umbilical cord of the stillborn puppy was tested for the presence of Hepatozoon spp. by PCR. The mother and the seven surviving puppies tested positive for H. canis by PCR at day 62 post-partum. Gamonts were detected in all dogs by buffy coat evaluation. Haematological and biochemistry results revealed mild abnormalities. In the stillborn puppy, spleen, umbilical cord, and amniotic fluid were positive for H. canis. CONCLUSION: The results confirm that vertical transmission is a possible route of H. canis infection in dogs, demonstrated by molecular detection of the pathogen in the stillborn puppy. In the seven surviving puppies, vertical transmission was the most likely transmission route. A potential impact of the level of parasitaemia on the health of puppies, as well as its pathogenesis, should be investigated further.

Ornithodoros (Pavlovskyella) ticks associated with a Rickettsia sp. in Pakistan.

BACKGROUND: Soft ticks (Ixodida: Argasidae) are medically important ectoparasites that mainly feed on birds and mammals, which play a key role in their geographic distribution and dispersion. Despite their importance, studies on soft ticks are scarce for many regions and countries of the world, including Pakistan. METHODS: In this study, 2330 soft ticks-179 larvae (7.7%), 850 nymphs (36.4%), 711 males (30.5%) and 590 females (25.3%)-were collected from animal shelters in 18 locations within five districts of Khyber Pakhtunkhwa, Pakistan. A subset of the collected ticks was processed for DNA extraction and polymerase chain reaction (PCR) for the amplification of tick 12S ribosomal DNA (rDNA), 16S rDNA and cytochrome c oxidase subunit I (cox1), and rickettsial 16S rDNA gene fragments. The obtained sequences were used for the construction of a phylogenetic tree. RESULTS: All the specimens were morphologically identified as Ornithodoros, and were morphologically similar to Ornithodoros tholozani. The genus was confirmed by sequencing partial 12S rDNA, 16S rDNA and cox1 gene fragments. Additionally, a Rickettsia sp. was detected in some of the collected ticks by PCR targeting 16S rDNA. The morphological relatedness of the tick specimens with O. tholozani was confirmed by phylogenetic analysis, in which the Ornithodoros sp. clustered with Ornithodoros tholozani and Ornithodoros verrucosus, both of which belong to the subgenus Pavlovskyella and have been previously reported from Israel, Ukraine and Iran. The phylogenetic tree also indicated that the Ornithodoros sp. from Pakistan corresponds to an undetermined species. Furthermore, the associated Rickettsia sp. grouped with the limoniae group of Rickettsia species previously reported from Argas japonicus ticks from China. CONCLUSIONS: This is the first molecular study of an Ornithodoros species from Pakistan. Further studies are essential to confirm its identity and possible pathogenicity with regard to its associated microorganisms in the studied region.

First report of Nosomma monstrosum ticks infesting Asian water buffaloes (Bubalus bubalis) in Pakistan.

Nosomma monstrosum Nuttall & Warburton, 1908 is a hard tick infesting mainly Asian water buffaloes, but it has also been recorded from cattle, horses, bears, dogs, wild boar and humans in the Oriental region. This tick species has previously not been recorded from Pakistan. A total of 15 adult N. monstrosum ticks (five females and ten males) were collected from an Asian water buffalo herd grazing in Haripur district, Khyber Pakhtunkhwa (KP), Pakistan. Molecular characterization using partial 12S, 16S rRNA, and cytochrome c oxidase 1 (cox1) gene sequences confirmed the identity as N. monstrosum. A phylogenetic analysis showed that N. monstrosum from Pakistan is closely related to specimens reported from Sri Lanka and Vietnam.

The Piroplasmida Babesia, Cytauxzoon, and Theileria in farm and companion animals: species compilation, molecular phylogeny, and evolutionary insights.

The order Piroplasmida, including the genera Babesia, Cytauxzoon, and Theileria is often referred to as piroplasmids and comprises of dixenous hemoprotozoans transmitted by ticks to a mammalian or avian host. Although piroplasmid infections are usually asymptomatic in wild animals, in domestic animals, they cause serious or life-threatening consequences resulting in fatalities. Piroplasmids are particularly notorious for the enormous economic loss they cause worldwide in livestock production, the restrictions they pose on horse trade, and the negative health impact they have on dogs and cats. Furthermore, an increasing number of reported human babesiosis cases are of growing concern. Considerable international research and epidemiological studies are done to identify existing parasite species, reveal their phylogenetic relationships, and develop improved or new drugs and vaccines to mitigate their impact. In this review, we present a compilation of all piroplasmid species, isolates, and species complexes that infect domestic mammals and which have been well defined by molecular phylogenetic markers. Altogether, 57 taxonomic piroplasmid entities were compiled, comprising of 43 piroplasmid species, 12 well-defined isolates awaiting formal species description, and two species complexes that possibly mask additional species. The extrapolation of the finding of at least 57 piroplasmid species in only six domestic mammalian groups (cattle, sheep, goat, horse, dog, and cat) allows us to predict that a substantially higher number of piroplasmid parasites than vertebrate host species exist. Accordingly, the infection of a vertebrate host species by multiple piroplasmid species from the same and/or different phylogenetic lineages is commonly observed. Molecular phylogeny using 18S rRNA genes of piroplasmids infecting domestic mammals results in the formation of six clades, which emerge due to an anthropocentric research scope, but not due to a possibly assumed biological priority position. Scrutinizing the topology of inferred trees reveals stunning insights into some evolutionary patterns exhibited by this intriguing group of parasites. Contrary to expectations, diversification of parasite species appears to be dominated by host-parasite cospeciation (Fahrenholz's rule), and, except for piroplasmids that segregate into Clade VI, host switching is rarely observed. When only domestic mammalian hosts are taken into account, Babesia sensu lato (s.l.) parasites of Clades I and II infect only dogs and cats, respectively, Cytauxzoon spp. placed into Clade III only infect cats, Theileria placed into Clade IV exclusively infect horses, wheras Theileria sensu stricto (s.s.) of Clade V infects only cattle and small ruminants. In contrast, Babesia s.s. parasites of Clade VI infect all farm and companion animal species. We outline how the unique ability of transovarial transmission of Babesia s.s. piroplasmids of Clade VI facilitates species diversification by host switching to other host vertebrate species. Finally, a deterioration of sequence fidelity in databases is observed which will likely lead to an increased risk of artifactual research in this area. Possible measures to reverse and/or avoid this threat are discussed.

Cultivation, cryopreservation and resuscitation of Theileria annulata transformed cells in serum-free media.

Introduction: Tropical theileriosis is a protozoan disease caused by Theileria annulata that affects cattle in Northern Africa, the Middle East and Asia where vector ticks of the genus Hyalomma occur. Various measures are applied to control the disease, including vaccination with attenuated T. annulata schizonts. Cultivation of T. annulata schizonts is mainly conducted in media containing Fetal Bovine Serum (FBS), which has some disadvantages such as costs, batch- to-batch variation and ethical concerns. Methods: In this study, we conducted three experiments to evaluate the ability of (1) T. annulata strains grown in RPMI with 10% FBS (RPMI-FBS) to adapt and grow in serum-free media (i.e., HL-1, RPMI without FBS supplementation, ISF-1, and M199), (2) a T. annulata strain grown in ISF-1 and subsequently frozen in this medium to grow in ISF-1 again after long-term storage in liquid nitrogen, and (3) a T. annulata strain freshly isolated from infected bovine lymphocytes to growin ISF-1, also after cryopreservation. Cell numbers, schizont index, the viability and generation doubling time were calculated in all experiments. Results and discussion: In the first experiment, the Hessiene and Beja cell lines from Tunisia previously cultivated in RPMI-FBS and adapted to serum-free media continued to grow significantly better in RPMI-FBS compared to the serum-freemedia. In the second experiment, a Tunisian cell line (Hessiene) cryopreserved in ISF-1 with 5%[v/v] dimethylsulfoxide (DMSO) grewbetter after thawing in RPMI-FBS compared to ISF-1 with a highly significant difference in cell growth (p < 0.001), whereas the third experiment showed that the Ankara cell line had similar growth characteristics in both RPMI-FBS and ISF-1 before and after thawing, with a shorter generation doubling time in ISF-1 than in RPMI-FBS (p = 0.23). Our findings suggest that freshly isolated cells can be propagated, frozen and thawed in serum-free media such as ISF-1, but once cells are adapted to cultivation in the presence of FBS or resuscitated from frozen storage, propagation in serum-free media may not perform as well as cultivation in RPMI-FBS.

Changes in the Ixodes ricinus microbiome associated with artificial tick feeding.

Artificial tick feeding systems (ATFS) can be used to study tick biology and tick-pathogen interactions. Due to the long feeding duration of hard ticks, antibiotics are commonly added to the in vitro blood meal to prevent the blood from decaying. This may affect the ticks' microbiome, including mutualistic bacteria that play an important role in tick biology. This effect was examined by the consecutive feeding of Ixodes ricinus larvae, nymphs, and adults in vitro with and without the supplementation of gentamicin and in parallel on calves. DNA extracted from unfed females was analyzed by 16S rRNA sequencing. The abundance of Candidatus Midichloria mitochondrii, Rickettsia helvetica and Spiroplasma spp. was measured by qPCR in unfed larvae, nymphs, and adults. Larvae and nymphs fed on calves performed significantly better compared to both in vitro groups. Adults fed on blood supplemented with gentamicin and B vitamins had a higher detachment proportion and weight compared to the group fed with B vitamins but without gentamicin. The detachment proportion and weights of females did not differ significantly between ticks fed on calves and in vitro with gentamicin, but the fecundity was significantly higher in ticks fed on calves. 16S rRNA sequencing showed a higher microbiome species richness in ticks fed on calves compared to ticks fed in vitro. A shift in microbiome composition, with Ca. Midichloria mitochondrii as dominant species in females fed as juveniles on calves and R. helvetica as the most abundant species in females previously fed in vitro was observed. Females fed in vitro without gentamicin showed significant lower loads of Ca. M. mitochondrii compared to females fed in vitro with gentamicin and ticks fed on calves. Spiroplasma spp. were exclusively detected in female ticks fed on cattle by qPCR, but 16S rRNA sequencing results also showed a low abundance in in vitro females exposed to gentamicin. In conclusion, the employed feeding method and gentamicin supplementation affected the ticks' microbiome composition and fecundity. Since these changes may have an impact on tick biology and vector competence, they should be taken into account in studies employing ATFS.

Molecular detection of Hepatozoon species infections in domestic cats living in Germany.

OBJECTIVES: Three species of protozoal Hepatozoon species (H felis, H canis and H silvestris) are known to infect cats in Europe. The objective of this study was to determine the prevalence of Hepatozoon species in samples from cats living in Germany that were submitted to a veterinary laboratory. METHODS: The study included cats tested for Hepatozoon species by PCR between 2007 and 2020 by the Laboklin laboratory. Travel history and haematological results were documented for cats with positive test results. From 2018 onwards, a partial 18S rRNA Hepatozoon gene fragment was sequenced from cats with positive PCR results. RESULTS: Sixty-four of 931 cats (7%) tested positive for Hepatozoon species. Sex and age did not have a statistically significant impact. Sequencing was carried out for 16 samples and revealed H felis in all cases. All cats with positive test results and a relevant travel history had been imported from the Mediterranean or south-eastern Europe. There were no autochthonous infections with Hepatozoon species. Leukocytosis, haemoconcentration and anaemia were the most common haematological abnormalities. CONCLUSIONS AND RELEVANCE: Although infections with Hepatozoon species in cats are usually subclinical, it may be useful to screen cats imported from the Mediterranean and south-eastern Europe for these pathogens to prevent local transmission cycles. There was no evidence of autochthonous infections in Germany; however, further investigations regarding a possible transmission of Hepatozoon species from infected cats to blood-feeding arthropods in Germany may be of interest. To avoid potential spread of the pathogens, ectoparasite prophylaxis is advisable.