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Piroplasmid parasites such as Theileria luwenshuni protozoa pose a global threat to both animal and human health. However, human theileriosis remains underexplored compared to infections caused by Plasmodium and Babesia species parasites. We investigated potential hemoparasite infections among 1,721 persons with fever, anemia, or both in Yunnan Province, China. Molecular detection identified 13 cases positive for T. luwenshuni protozoa, of which 5 patients were further confirmed by Western blot antibody analysis. We also identified 6 babesiosis cases, 3 infections with B. microti and 3 with novel Babesia spp. Subsequent vector and host investigations in the vicinity of the index cases revealed T. luwenshuni protozoa in 1 tick and 53 livestock animals. Of note, 3.3% combined vector-host samples tested positive for genetically diverse Babesia species. Our findings highlight the endemic circulation of T. luwenshuni and Babesia spp. parasites in southwest China, underscoring their importance as emerging public health concerns.

Tropical theileriosis, caused by the protozoan parasite Theileria annulata and transmitted by several species of ixodid ticks of the genus Hyalomma, is an economically important disease of bovines. Concerningly, studies conducted in recent years have shown an increase in the rate of failure when using the primary drug of treatment, buparvaquone (BPQ), particularly in infection caused by T. annulata populations bearing V135A and P253S mutations on the Cytochrome b ( Cyto b ) gene of the parasite mitochondrial genome. The aim of this study was to demonstrate the relationship between BPQ-resistance and V135A and P253S mutations utilising an in vivo experimental set-up and to assess the tick transmissibility of drug-resistant populations. Additionally, the pharmacokinetics of BPQ in healthy and infected calves were compared to evaluate any relationship between plasma drug concentration and treatment failure. The study results demonstrated that, despite four consecutive BPQ treatments, animals infected with the resistant isolates exhibited more severe clinical signs, including longer periods of pyrexia, longer periods of schizont and piroplasm parasitemia, and the death of one animal. In addition, 3-(4,5-dimethylthiazol--yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that all cell lines derived from animals infected with the mutant genotypes exhibited resistance to high BPQ concentrations. Unexpectedly, despite substantial calf-to-calf variation during the experiment, the genetic structure of the parasite population remained largely unchanged and no strong evidence for a major genotypic shift was detected. Plasma BPQ levels were similar in all groups tested. There was no association between plasma concentrations of BPQ and parasitological or clinical response to treatment. Live parasitaemia was observed even at high plasma BPQ levels in animals infected with resistant isolates. Significantly, drug resistant parasite populations harbouring either V135A or P253S mutations was transferred between the host and vector ticks, indicating the potential for resistant parasites to be transmitted from cattle in the field, thereby facilitating their maintenance in natural populations.

Background Infections with Babesia bovis , Babesia bigemina, Theileria species and Anaplasma marginale are endemic in Kenya yet there is a lack of adequate information on their genotypes. This study established the genetic diversities of the above tick-borne hemoparasites infecting cattle in Kenya. Methods Nested PCR and sequencing were used to determine the prevalence and genetic diversity of the above parasites in 192 cattle blood samples collected from Ngong and Machakos farms. B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein 1a, A. marginale major surface protein 5, Theileria spp. 18S rRNA, T. parva p104 and T. orientalis major piroplasm surface protein were used as the marker genes. Results B. bovis , B. bigemina , T. parva , T. velifera , T. taurotragi , T. mutans and A. marginale were prevalent in both farms, whereas T. ovis, Theileria sp . (buffalo) and T. orientalis were found only in Ngong farm. Co-infections were observed in more than 50 % of positive samples in both farms. Babesia parasites and A. marginale sequences were highly conserved while T. parva and T. orientalis were polymorphic. Cattle-derived T. parva was detected in Machakos farm. However, cattle and buffalo–derived Theileria were detected in Ngong farm suggesting interactions between cattle and wild buffaloes. Generally, the pathogens detected in Kenya were genetically related to the other African isolates but different from the isolates in other continents. Conclusions The current findings reaffirm the endemicity and co-infection of cattle with tick-borne hemoparasites, and the role of wildlife in pathogens transmission and population genetics in Kenya.

In southern African transfrontier conservation areas (TFCAs), people, livestock and wildlife share space and resources in semi-arid landscapes. One consequence of the coexistence of wild and domestic herbivores is the risk of pathogen transmission. This risk threatens local livelihoods relying on animal production, public health in the case of zoonoses, national economies in the context of transboundary animal diseases, and the success of integrated conservation and development initiatives. The level of interaction between sympatric wild and domestic hosts, defining different wildlife/livestock interfaces, characterizes opportunities of pathogen transmission between host populations. Exploring the relationship between infection burden and different types of wildlife/domestic interfaces is therefore necessary to manage the sanitary risk in animal populations through control options adapted to these multi-host systems. Here, we assessed the infection burdens of sympatric domestic cattle (Bos taurus/Bos indicus) and African buffalo (Syncerus caffer) at an unfenced interface and compared the infection burdens of cattle populations at different wildlife/livestock interfaces in the Great Limpopo TFCA. Patterns of infection in ungulate populations varied between wild and domestic hosts and between cattle populations at different wildlife/livestock interfaces. Foot-and-mouth disease, Rift Valley fever and theileriosis infections were detected in buffalo and cattle at unfenced interfaces; bovine tuberculosis was only present in buffalo; and brucellosis and lumpy skin disease only in cattle. At unfenced interfaces, cattle populations presented significantly higher Theileria parva and brucellosis prevalence. We hypothesize that cattle populations at wildlife/livestock interfaces face an increased risk of infection compared to those isolated from wildlife, and that the type of interface could influence the diversity and quantity of pathogens shared. Additional host behavioural and molecular epidemiological studies need to be conducted to support this hypothesis. If it is confirmed, the management of wildlife/livestock interfaces will need to be considered through the prism of livestock and public health.

Babesia ovis is a tick-transmitted intraerythrocytic apicomplexan parasite that causes ovine babesiosis, a disease with high economic impact in endemic regions. Although Rhipicephalus bursa is a known biological vector of B. ovis , the dynamics of vertical (transovarial) transmission are poorly understood. In the present study, the transovarial transmission of B. ovis was investigated experimentally in four continuous generations of R. bursa under laboratory controlled conditions. A total of 11 sheep were used during the study. An initial stabilate co-infected with B. ovis and Theileria ovis was inoculated into a splenectomized sheep, followed by an infestation with Babesia- free adult R. bursa . Study of dead tick bodies and larval pools by molecular methodology confirmed the elimination of T. ovis and the vertical transmission B. ovis . Rhipicephalus bursa ticks derived vertically from F 1 to F 4 generations kept high levels of infection (98% in F 3 adults), and consistently led to severe clinical babesiosis in both immunosuppressed and immunocompetent sheep. Although all feeding stages became infected while feeding on their hosts, only adult ticks were able to transmit the parasite to vertebrate hosts. Our findings demonstrate that B. ovis can be transmitted vertically through several tick generations, while preserving its ability to cause severe disease, even without selective pressure. The virulent, mono-infected B. ovis strain developed in this study will provide an infectious challenge model for future vaccine and pathogenesis studies under field-relevant conditions. Babesia ovi s est un parasite apicomplexe intraérythrocytaire transmis par les tiques et responsable de la babésiose ovine, une maladie à fort impact économique dans les régions endémiques. Bien que Rhipicephalus bursa soit un vecteur biologique connu de B. ovis , la dynamique de la transmission verticale (transovarienne) est mal comprise. Dans la présente étude, la transmission transovarienne de B. ovis a été étudiée expérimentalement sur quatre générations continues de R. bursa dans des conditions contrôlées en laboratoire. Au total, 11 moutons ont été utilisés au cours de l’étude. Un stabilat initial co-infecté par B. ovis et Theileria ovis a été inoculé à un mouton splénectomisé, suivi d’une infestation par des adultes de R. bursa indemnes de Babesia . L’étude des tiques mortes et des lots de larves par méthodologie moléculaire a confirmé l’élimination de T. ovis et la transmission verticale de B. ovis . Les tiques R. bursa dérivées verticalement des générations F 1 à F 4 ont conservé des niveaux d’infection élevés (98 % chez les adultes F 3 ) et ont systématiquement entraîné une babésiose clinique sévère chez les moutons immunodéprimés et immunocompétents. Bien que tous les stades d’alimentation aient été infectés en se nourrissant de leurs hôtes, seules les tiques adultes ont pu transmettre le parasite à leurs hôtes vertébrés. Nos résultats démontrent que B. ovis peut être transmis verticalement par plusieurs générations de tiques tout en préservant sa capacité à provoquer une maladie grave, même en l’absence de pression sélective. La souche virulente et mono-infectée de B. ovis développée dans cette étude fournira un modèle infectieux pour de futures études de vaccin et de pathogénèse dans des conditions de terrain.

The prevalence of Theileria spp. infecting sheep and goats were investigated in two cities and surroundings in northwest Saudi Arabia. Blood samples from 403 sheep and goats from Madina ( n = 201) and Tabuk ( n = 202) cities (177 from sheep and 226 from goats) were investigated. Blood samples were examined microscopically for the presence of intraerythrocytic bodies suggestive of Theileria as well as they were investigated using molecular techniques. DNA was extracted from blood and ticks and subjected to polymerase chain reaction amplification using specific primers. The primers used amplified a fragment of the 18S rRNA region (450 bp) targeting the hypervariable region IV. A total of 63 ticks belonging to five species were collected from sheep and goats for determination of their involvement of lifecycle of Theileria . Ticks were identified morphologically and confirmed molecularly utilizing cytochrome oxidase C subunit 1 gene (COXI) amplification. The results indicated that: microscopic examination revealed 24 (6%) of the samples investigated showed intraerythrocytic bodies suggestive of Theileria . Positive samples were only obtained from sheep whereas goats yielded negative results. A total of 33 (18.6%) sheep samples were positive for Theileria infection using polymerase chain reaction targeting the partial 18S rDNA and DNA sequencing. Theileria infection was more prevalent in animals that were less than 2 years of age compared with older animals. There was no difference in the prevalence of the infection between male and female sheep in both cities. All positive sheep were detected during the summer and none of the samples collected during the winter were positive. Phylogenetic analysis revealed that the sequences obtained from Theileria species reported in the present study grouped with sequences from Theileria ovis from different countries. Ticks were identified as Hyalomma dromedarii , Hyalomma, marginatum , Hyalomma impeltatum and Hyalomma anatolicum anatolicum . T. ovis DNA was detected from Hyalomma dromedarii and Hyalomma impeltatum suggesting that they are potential vectors of this piroplasm in sheep from Madina and Tabuk cities. This report is considered the first report of T. ovis infecting sheep from Madina and Tabuk, furthermore, it is the first report determining the vectors responsible for transmission of the infection in sheep in northwest Saudi Arabia. The data generated from this study will undoubtedly pave the way for the detection and control of ovine and caprine theileriosis in Madina and Tabuk regions.

Arthropod-borne apicomplexan pathogens that cause asymptomatic persistent infections present a significant challenge due to their life-long transmission potential. Although anti-microbials have been used to ameliorate acute disease in animals and humans, chemotherapeutic efficacy for apicomplexan pathogen elimination from a persistently infected host and removal of transmission risk is largely unconfirmed. The recent re-emergence of the apicomplexan Theileria equi in U.S. horses prompted testing whether imidocarb dipropionate was able to eliminate T. equi from naturally infected horses and remove transmission risk. Following imidocarb treatment, levels of T. equi declined from a mean of 104.9 organisms/ml of blood to undetectable by nested PCR in 24 of 25 naturally infected horses. Further, blood transfer from treated horses that became nested PCR negative failed to transmit to naı¨ve splenectomized horses. Although these results were consistent with elimination of infection in 24 of 25 horses, T. equi-specific antibodies persisted in the majority of imidocarb treated horses. Imidocarb treatment was unsuccessful in one horse which remained infected as measured by nested PCR and retained the ability to infect a naı¨ve recipient via intravenous blood transfer. However, a second round of treatment eliminated T. equi infection. These results support the utility of imidocarb chemotherapy for assistance in the control and eradication of this tick-borne pathogen. Successful imidocarb dipropionate treatment of persistently infected horses provides a tool to aid the global equine industry by removing transmission risk associated with infection and facilitating international movement of equids between endemic and non-endemic regions.

We describe for the first time the detection of Coxiella-like bacteria (CLB), Theileria luwenshuni, and T. ovis endosymbionts in blood-sucking deer keds. Eight deer keds attached to a Korean water deer were identified as Lipoptena fortisetosa (Diptera: Hippoboscidae) by morphological and genetic analyses. Among the endosymbionts assessed, CLB, Theileria luwenshuni, and T. ovis were identified in L. fortisetosa by PCR and nucleotide sequencing. Based on phylogeny, CLB 16S rRNA sequences were classified into clade B, sharing 99.4% identity with CLB from Haemaphysalis longicornis in South Korea. Although the virulence of CLB to vertebrates is still controversial, several studies have reported clinical symptoms in birds due to CLB infections. The 18S rRNA sequences of T. luwenshuni and T. ovis in this study were 98.8-100% identical to those in GenBank, and all of the obtained sequences of T. ovis and T. luwenshuni in this study were 100% identical to each other, respectively. Although further studies are required to positively confirm L. fortisetosa as a biological vector of these pathogens, strong genetic relationships among sequences from this and previous studies suggest potential transmission among mammalian hosts by ticks and keds.

Background East Coast fever (ECF) is a devastating disease of cattle and a significant constraint to improvement of livestock production in sub-Saharan Africa. The protozoan parasite causing ECF, Theileria parva , undergoes obligate sexual stage development in its tick vector Rhipicephalus appendiculatus . Tick-borne acquisition and transmission occurs transstadially; larval and nymphal ticks acquire infection while feeding and transmit to cattle when they feed after molting to the next stage. Much of the current knowledge relating to tick-borne acquisition and transmission of T. parva has been derived from studies performed during acute infections where parasitemia is high. In contrast, tick-borne transmission during the low-level persistent infections characteristic of endemic transmission cycles is rarely studied. Methods Cattle were infected with one of two stocks of T. parva (Muguga or Marikebuni). Four months post-infection when parasites were no longer detectable in peripheral blood by PCR, 500 R. appendiculatus nymphs were fed to repletion on each of the cattle. After they molted to the adult stage, 20 or 200 ticks, respectively, were fed on two naïve cattle for each of the parasite stocks. After adult ticks fed to repletion, cattle were tested for T. parva infection by nested PCR and dot blot hybridization. Results Once they had molted to adults the ticks that had fed as nymphs on Muguga and Marikebuni infected cattle successfully transmitted Theileria parva to all naïve cattle, even though T. parva infection was not detectable by nested PCR on salivary gland genomic DNA of a sample of individual ticks. However, a salivary gland homogenate from a single Marikebuni infected tick was able to infect primary bovine lymphocytes. Infection was detected by nested p104 PCR in 3 of 4 calves and detected in all 4 calves by T. parva 18S nested PCR/dot blot hybridization. Conclusion We show that R. appendiculatus ticks are able to acquire T. parva parasites from infected cattle even in the absence of detectable parasitemia. Although infection was undetectable in a sample of individual ticks, cumulatively as few as 20 ticks were able to transmit T. parva to naïve cattle. These results have important implications for our understanding of T. parva transmission by R. appendiculatus in ECF endemic regions.

The establishment of good experimental models for Theileria sp. infection is important for theileriosis research. Routinely, infection of ticks is accomplished by feeding on parasite-infected animals (sheep, cows and horses), which raises practical and ethical problems, driving the search for alternative methods of tick infection. Artificial tick feeding systems are based mainly on rearing ticks on host-derived or hand-made artificial membranes. We developed a modified feeding assay for infecting nymphal stages of Hyalomma anatolicum ticks with Theileria lestoquardi, a highly pathogenic parasite of sheep. We compared two different membranes: an artificial silicone membrane and a natural alternative using mouse skin. We observed high attachment rates with mouse skin, whereas in vitro feeding of H. anatolicum nymphs on silicone membranes was unsuccessful. We could infect H. anatolicum nymphs with T. lestoquardi and the emerging adult ticks transmitted infective parasites to sheep. In contrast, similar infections with Rhipicephalus bursa, a representative tick with short mouth-parts that was proposed as a vector for T. lestoquardi, appeared not to be a competent vector tick species. This is the first report of an experimentally controlled infection of H. anatolicum with T. lestoquardi and opens avenues to explore tick-parasite dynamics in detail.