Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Vector competence is a component of vectorial capacity and depends on genetic determinants affecting the ability of a vector to transmit a pathogen. These determinants affect traits such as tick-host-pathogen and susceptibility to pathogen infection. Therefore, the elucidation of the mechanisms involved in tick-pathogen interactions that affect vector competence is essential for the identification of molecular drivers for tick-borne diseases. In this review, we provide a comprehensive overview of tick-pathogen molecular interactions for bacteria, viruses, and protozoa affecting human and animal health. Additionally, the impact of tick microbiome on these interactions was considered. Results show that different pathogens evolved similar strategies such as manipulation of the immune response to infect vectors and facilitate multiplication and transmission. Furthermore, some of these strategies may be used by pathogens to infect both tick and mammalian hosts. Identification of interactions that promote tick survival, spread, and pathogen transmission provides the opportunity to disrupt these interactions and lead to a reduction in tick burden and the prevalence of tick-borne diseases. Targeting some of the similar mechanisms used by the pathogens for infection and transmission by ticks may assist in development of preventative strategies against multiple tick-borne diseases.

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The genus Anaplasma (Rickettsiales: Anaplasmataceae) comprises obligate intracellular Gram-negative bacteria that are mainly transmitted by ticks, and currently includes six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma infections are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC ("European Network of Neglected Vectors and Vector-Borne Diseases"). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and vertebrates and their application.

Babesiosis is a tick borne disease (TBD) caused by parasites of the genus Babesia, with 13 considerable worldwide economic, medical, and veterinary impact. Babesiosis and other 14 TBDs were considered responsible for 50% of the deaths of cattle occurred in 15Mozambique in the first year after importation from neighbouring countries. 16Here, we present the detection of B. bigemina and B. bovis in cattle from Mozambique 17 and the development of a novel PCR method. For this study, blood samples were 18 collected in one farm located near Maputo city. The samples were analyzed using a 19 previously described nested PCR and a novel hot-start PCR method. Primers were 20 selected for the hot-start PCR based on the putative gene of an undescribed aspartic 21 protease named babesipsin, present in both B. bovis and B. bigemina. The combination of 22 hot start polymerase and long primers (29-31bp) were in this study determinant for the 23 Manuscript 2 successful amplification and detection in only one PCR. The babesipsin hot-start PCR is 24 a fast, simple, and sensitive method, with some advantages over current methods. A total 25of 117 field samples were tested by hot-start PCR, and 90 were positive for B. bigemina 26 (77%), 82 were positive for B. bovis (70%), 61 were mixed infections (52%) and only 6 27 were negative (5.1%). The results confirm that this area of Mozambique is endemic for 28 Babesiosis, and that this TBD should be regarded as a threat for imported cattle. 29 30

Ticks are among the most prevalent blood-feeding arthropods, and they act as vectors and reservoirs for numerous pathogens. Sialotranscriptomic characterizations of tick responses to blood feeding and pathogen infections can offer new insights into the molecular interplay occurring at the tick-host-pathogen interface. In the present study, we aimed to identify and characterize Rhipicephalus bursa salivary gland (SG) genes that were differentially expressed in response to blood feeding and Babesia ovis infection. Our experimental approach consisted of RNA sequencing of SG from three different tick samples, fed-infected, fed-uninfected, and unfed-uninfected, for characterization and inter-comparison. Overall, 7,272 expressed sequence tags (ESTs) were constructed from unfed-uninfected, 13,819 ESTs from fed-uninfected, and 15,292 ESTs from fed-infected ticks. Two catalogs of transcripts that were differentially expressed in response to blood feeding and B. ovis infection were produced. Four genes coding for a putative vitellogenin-3, lachesin, a glycine rich protein, and a secreted cement protein were selected for RNA interference functional studies. A reduction of 92, 65, and 51% was observed in vitellogenin-3, secreted cement, and lachesin mRNA levels in SG, respectively. The vitellogenin-3 knockdown led to increased tick mortality, with 77% of ticks dying post-infestation. The reduction of the secreted cement protein-mRNA levels resulted in 46% of ticks being incapable of correctly attaching to the host and significantly lower female weights post-feeding in comparison to the control group. The lachesin knockdown resulted in a 70% reduction of the levels associated with B. ovis infection in R. bursa SG and 70% mortality. These results improved our understanding of the role of tick SG genes in Babesia infection/proliferation and tick feeding. Moreover, lachesin, vitellogenin-3, and secreted cement proteins were validated as candidate protective antigens for the development of novel tick and tick-borne disease control measures.

A series of artemisinin-vinyl sulfone hybrid molecules with the potential to act in the parasite food vacuole via endoperoxide activation and falcipain inhibition was synthesized and screened for antiplasmodial activity and falcipain-2 inhibition. All conjugates were active against the Plasmodium falciparum W2 strain in the low nanomolar range and those containing the Leu-hPhe core inhibited falcipain-2 in low micromolar range. Keywordsantimalarial; artemisinin; FP-2; Vinylsulfone Artemisinin, 1, a sesquiterpene lactone isolated from the Artemisia annua Chinese herb, and its analogues (e.g. artemether, 2, arteether, 3, and artesunate, 4) were a major breakthrough in malaria chemotherapy because they produce a very rapid therapeutic response, particularly against multidrug-resistant Plasmodium falciparum malaria. 1,2 Despite the rapid clearance of parasites, the short half-lives of these compounds lead to many late recrudescences after monotherapy. 3 Thus, artemisinin-based combination therapy (ACT) has now been recommended by the World Health Organisation as standard therapy for falciparum malaria. 4 Cysteine proteases from malaria parasites are of particular interest as therapeutic targets due to their role in parasite development. 5 P. falciparum expresses four cysteine proteases from the papain family known as falcipains, of which falcipain-2 (FP-2) 6,7 and falcipain-3 (FP-3) 7,8 are the most relevant as therapeutic targets. Peptidyl vinyl sulfones, e.g. 5, are potent irreversible inhibitors of falcipains, acting as Michael acceptors of the catalytic cysteine residue. 9 Falcipain inhibitors have been shown to inhibit the development of cultured erythrocytic parasites by blocking the hydrolysis of host hemoglobin and to cure mice infected with lethal malaria infections. 10 *Corresponding authors: Phone: +351217946476; Fax: +35 1217946470; fclopes@ff.ul.pt. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA concern regarding the use of protease inhibitors as antimalarials is that selection of drugresistant mutants will eventually occur. Indeed, parasites resistant to a dipeptidyl vinyl sulfone have been selected in the laboratory, although this resistance was somewhat unstable. 11 Thus, dipeptidyl vinyl sulfones are obvious candidates for combination antimalarial therapy as a strategy to retard the development of resistance. This information prompted us to design artemisinin-vinyl sulfone hybrid molecules with the potential to help prevent multi-drug resistance in P. falci...