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Purpose: Tick-borne disease (TBD) prevention and control is dependent on a thorough understanding of disease vectors and pathogens. In Malawi, there is little information on molecular investigations of cattle tick-borne pathogens (TBPs). This study aimed to detect TBPs infecting cattle in southern Malawi which has the highest cattle population in Malawi. Materials and Methods: A total of 220 apparently healthy cattle blood samples were collected in 6 southern Malawi districts. Detection of TBPs was done using polymerase chain reaction (PCR) tests with species-specific primers. Results: A 72.3% overall infection rate (159/220) was recorded, with Anaplasma marginale (51/220, 23.2%), A. bovis (21/220, 9.6%), A. phagocytophilum (49/220, 22.3%), Babesia bigemina (76/220, 34.5%), B. bovis (16/220, 7.3%), B. naoakii(6/220, 2.7%), Theileria parva (34/220, 15.5%), T. mutans, (9/220, 4.1%), and T. taurotragi (49/220, 22.3%) as the detected pathogens. Conclusion: The study reports B. naoakii, and B. bovis for the first time in cattle from Malawi. The study provides essential information on the distribution of TBDs and for carrying out potential control efforts in Malawi.

The present study aimed to evaluate the anti-Babesia effect of MMV390048, a drug that inhibits Plasmodium by targeting the phosphatidylinositol 4-kinase (PI4K). The half inhibitory concentration (IC(50)) of MMV390048 against the in vitro growth of Babesia gibsoni was 6.9 ± 0.9 μM. In immunocompetent mice, oral treatment with MMV390048 at a concentration of 20 mg/kg effectively inhibited the growth of B. microti (Peabody mjr strain). The peak parasitemia in the control group was 30.5%, whereas the peak parasitemia in the MMV390048-treated group was 3.4%. Meanwhile, MMV390048 also showed inhibition on the growth of B. rodhaini (Australia strain), a highly pathogenic rodent Babesia species. All MMV390048-treated mice survived, whereas the mice in control group died within 10 days postinfection (DPI). The first 7-day administration of MMV390048 in B. microti-infected, severe combined immunodeficiency (SCID) mice delayed the rise of parasitemia by 26 days. Subsequently, a second 7-day administration was given upon recurrence. At 52 DPI, a parasite relapse (in 1 out of 5 mice) and a mutation in the B. microti PI4K L746S, a MMV390048 resistance-related gene, were detected. Although the radical cure of B. microti infection in immunocompromised host SCID mice was not achieved, results from this study showed that MMV390048 has excellent inhibitory effects on Babesia parasites, revealing a new treatment strategy for babesiosis: targeting the B. microti PI4K.

Bovine babesiosis causes morbidity in tropical and subtropical countries worldwide. The present study aimed to determine the seroprevalence of Babesia bigemina and B. bovis in cattle and water buffaloes in Menoufia province, where the second-highest population of bovines in Lower Egypt are raised. A total of 506 blood samples were collected from cattle (N = 262) and water buffaloes (N = 244) in Menoufia province, Egypt. Seroprevalences of B. bigemina and B. bovis in the samples were determined using recombinant Babesia antigen-specific enzyme-linked immunosorbent assays (ELISA). In cattle, the seroprevalences of B. bigemina and B. bovis were 41.60 and 38.17% (37.40 and 35.88% for IgM and 9.54 and 6.11% for IgG), respectively, whereas those of water buffaloes were 35.66 and 31.97% (27.87 and 21.72% for IgM and 15.16 and 15.16% for IgG), respectively. Statistically significant changes in the seroprevalences of the two infective agents were recorded on the basis of region and season of sample collection. In conclusion, babesiosis is frequent and presents a threat of an epidemic among bovines in Menoufia province. In turn, control of bovine babesiosis is required because of its potential to detrimentally affect milk and meat production in Menoufia province.

Tick-borne diseases mainly, theileriosis, babesiosis and anaplasmosis cause significant economic losses in livestock globally, including Turkey. The tick-borne pathogens of small ruminants in Turkey have been studied widely but information on molecular characterization and disease occurrence is still limited. In this study, both microscopy and molecular detection and characterization for Theileria spp. Babesia ovis, Anaplasma ovis and Anaplasma phagocytophilum was conducted. A total of 133 blood samples of tick-infested small ruminants (105 sheep and 28 goats) were collected from Turkey: half of the animals had clinical signs of tick-borne disease infections. Using PCR assays and microscopy, 90.2% and 45.1% of the samples were positive for at least one pathogen, respectively. Overall, the infection rates of A. phagocytophilum, B. ovis, A. ovis, Theileria spp. were 66.7%, 62.4%, 46.6% and 7.0%, respectively. Fifty-nine of the 133 (44.4%) samples were co-infected with two or more pathogens. Sex, season and B. ovis positivity were significant risk factors for occurrence of clinical disease. Sequence and phylogenetic analysis based on B. ovis 18S small subunit rRNA, A. ovis major surface protein 4, Theileria spp. 18S rRNA and A. phagocytophilum 16S rRNA genes showed that the isolates in this study clustered together in well-supported clades with those previously collected from Turkey and other countries. The study shows B. ovis as the most significant pathogen associated with clinical and fatal cases in small ruminants from Turkey. Female sex and summer season are associated with increased risk of the disease. This study shows high infection rates with the pathogens among small ruminants including A. phagocytophilum which has veterinary and public health importance.

In the present study, we screened 502 natural product compounds against the in vitro growth of Babesia (B.) bovis. Then, the novel and potent identified compounds were further evaluated for their in vitro efficacies using viability and cytotoxicity assays. The in vivo inhibitory effects of the selected compounds were evaluated using B. microti “rodent strain” in mice model. Three potent compounds, namely, Rottlerin (RL), Narasin (NR), Lasalocid acid (LA), exhibited the lowest IC50 (half-maximal inhibitory concentration) as follows: 5.45 ± 1.20 μM for RL, 1.86 ± 0.66 μM for NR, and 3.56 ± 1.41 μM for LA. The viability result revealed the ability of RL and LA to prevent the regrowth of treated parasite at 4 × IC50 and 2 × IC50, respectively, while 4 × IC50 of NR was sufficient to stop the regrowth of parasite. The hematology parameters of B. microti in vivo were different in the NR-treated groups as compared to the infected/untreated group. Interestingly, intraperitoneal administration of NR exhibiting inhibition in the growth of B. microti in mice was similar to that observed after administration of the commonly used antibabesial drug, diminazene aceturate (DA) (76.57% for DA, 74.73% for NR). Our findings indicate the richness of natural product compounds by novel potent antibabesial candidates, and the identified potent compounds, especially NR, might be used for the treatment of animal babesiosis.

Background: Drug resistance and severe side effects are major challenges in the treatment of babesiosis as they lead to less choices for treatment. Development of new drugs to enrich the treatment strategies and delay the emergence of drug resistance in parasites is still needed. Naphthoquine (NQ) combined with artemisinin treats Plasmodium infection by rapid parasite clearance. The current study repurposed NQ as a babesiosis drug treatment by evaluating the effects of naphthoquine phosphate (NQP) as a single dose treatment for babesiosis. Methods: In vitro anti-Babesia activity of NQP was tested on Babesia gibsoni cultures. The inhibition of parasite growth was verified using a SYBR green I-based fluorescence assay. In vivo efficacy of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored. Results: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3 ± 0.5 μM. Oral administration of NQP for 5 successive days at a dose of 40 mg/kg of body weight resulted in significant inhibition on parasite growth compared with the control group. All mice in NQP-treated group survived, whereas the mice in control group died between days 6 and 9 post infection. Conclusion: This is the first study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. The results showed that NQP is a promising drug for babesiosis treatment and drug repurposing may provide new treatment strategies for babesiosis.

5-aminolevulenic acid (ALA), an endogenous, non-proteinogenic, naturally occurring amino acid found in diverse organisms, is a precursor of heme biosynthesis. For apicomplexan protozoan parasites, an ALA and sodium ferrous citrate (SFC) combination was previously evaluated and suggested as a potential drug candidate for Plasmodium falciparum malaria. This study aimed to evaluate the potential of this combination against bovine babesiosis. ALA administration at 100 and 500 µM coupled with 10 µM SFC in culture medium significantly inhibited intraerythrocytic development and growth of Babesia bovis, which causes cerebral babesiosis in cattle, under in vitro culture. However, administration of 10 µM SFC only in the medium did not inhibit parasite growth. ALA/SFC was efficacious in treating babesiosis in an experimental animal model with B. microti, which causes debilitating babesiosis in mice. Female BALB/c mice were infected with B. microti and administered a single oral dose of ALA/SFC combination daily in different concentrations for 30 days. Treatment with ALA/SFC at 4/0.4 mg/kg body weight significantly suppressed parasite development in the mice blood circulation and resulted in significantly lower parasitemia. Moreover, body weight loss in the mice has been improved significantly compared with the control group at the peak of parasitemia. Treated mice showed moderate decreases in red blood cell count, hemoglobin value, and hematocrit compared with those observed in the control group, indicating an effect in moderating progressive anemia. These findings suggested the potential of ALA/SFC to achieve symptomatic improvement against bovine babesiosis.

Tick-borne diseases (TBD) cause enormous losses for farmers. Backyard raising comprises majority of the livestock population in the Philippines, but TBD information in backyard livestock is scarce. In this study, 48 cattle and 114 water buffalo samples from Quezon province, Philippines were molecularly screened for tick-borne pathogens. Anaplasma marginale (16.67%) and hemoplasma (20.99%) were detected in the samples. A. marginale infection (P=0.0001) was significantly higher in cattle, while hemoplasma infection (P=0.011) was significantly higher in water buffaloes. A. marginale isolates from this study were highly similar to previous isolates from the Philippines while Mycoplasma wenyonii and Candidatus Mycoplasma haemobos were the identified hemoplasma species. Our findings reveal additional information on the TBD situation of Philippine backyard livestock.

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4–99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.

Sika deer (Cervus nippon) is widely distributed in Asian countries and is one of the most common wildlife animals in Hokkaido, Japan. Previous studies identified Theileria spp. in sika deer in Japan including Theileria sp. Thrivae belonging to T. cervi group and Theileria sp. sola belonging to T. capreoli group. However, the studies failed to differentiate these two species without sequencing. Therefore, epidemiological information on cervine theileriosis in Hokkaido, Japan is limited. This study differentiated the two Theileria spp. using restriction fragments length polymorphism (RFLP). Based on the PCR-RFLP, Theileria spp. were identified in 103 (88.0%) of 117 samples, and the prevalence of each parasites were 86.3% (n = 101) and 57.3% (n = 67) for Theileria sp. Thrivae and T. capreoli-like, respectively. Phylogenetic analysis based on the 18S rRNA showed a close relationship between Theileria sp. Thrivae and T. cervi in China. In addition, phylogenetic analysis of internal transcribed spacer regions also showed a close relationship between Theileria sp. Thrivae and T. cervi.

Information regarding the prevalence and genetic characterization of Anaplasma spp., Babesia spp. and Theileria spp. infecting sheep in Qinghai province, northwestern China, is limited. In this study, 277 sheep blood samples were collected from 7 counties in Qinghai and tested by PCR to detect and identify the above mentioned tick-borne pathogens. Among the 277 sheep, 176 (63.5%) were infected with at least one pathogen. A. ovis was the most prevalent pathogen (n = 112, 40.4%), followed by T. luwenshuni (n = 108, 39.0%), T. uilenbergi (n = 65, 23.5%) and B. motasi sp. L/N/T (n = 11, 4.0%). Interestingly and importantly, 83 (30.0%) infections with two or more pathogens were identified. The analysis of the nucleotide sequences of targeted genes showed that A. ovis msp4, B. motasi sp. L/N/T rap-1b, T. luwenshuni 18S rRNA and T. uilenbergi 18S rRNA sequences from Qinghai were not conserved. These sequences shared 97.7–99.7%, 87.0–99.8%, 98.2–100% and 91.3–100% identities with previously published isolates, respectively. The phylogenetic analysis revealed that all A. ovis msp4 sequences belonged to the same clade. On the other hand, B. motasi sp. L/N/T rap-1b isolates showed a closer relationship with B. motasi sp. BQ1/Lintan. One T. luwenshuni and one T. uilenbergi sequence from this study formed branches divergent from the remaining T. luwenshuni and T. uilenbergi isolates. Overall, these results provide a better understanding of the prevalence and molecular characterization of TBPs in sheep in Qinghai and suggest that these pathogens require more attention from veterinarians.

Due to drug resistance, commonly used anti-Babesia drugs have limited efficacy against babesiosis and inflict severe side effects. Tafenoquine (TAF) was approved by the U.S. Food and Drug Administration in 2018 for the radical cure of Plasmodium vivax infection and for malaria prophylaxis. Here, we evaluated the efficacy of TAF for the treatment of Babesia infection and elucidated the suspected mechanisms of TAF activity against Babesia parasites. Parasitemia and survival rates of Babesia rodhaini-infected BALB/c and SCID mice were used to explore the role of the immune response in Babesia infection after TAF treatment. Parasitemia, survival rates, body weight, vital signs, complete blood count, and blood biochemistry of B. gibsoni-infected splenectomized dogs were determined to evaluate the anti-Babesia activity and side effects of TAF. Then, to understand the mechanism of TAF activity, hydrogen peroxide was used as an oxidizer for short-term B. rodhaini incubation in vitro, and the expression levels of antioxidant enzymes were confirmed using B. microti-infected mice by reverse transcription-quantitative PCR (qRT-PCR). Acute B. rodhaini and B. gibsoni infections were rapidly eliminated with TAF administration. Repeated administration of TAF or a combination therapy with other antibabesial agents is still needed to avoid a potentially fatal recurrence for immunocompromised hosts. Caution about hyperkalemia should be taken during TAF treatment for Babesia infection. TAF possesses a babesicidal effect that may be related to drug-induced oxidative stress. Considering the lower frequency of glucose-6-phosphate dehydrogenase deficiency in animals compared to that in humans, TAF use on Babesia-infected farm animals and pets is eagerly anticipated.

An enzyme-linked immunosorbent assay (ELISA) based on recombinant SAG1-related sequence 2 of Toxoplasma gondii (rTgSRS2) was developed to detect toxoplasmosis in cats. The specificity and sensitivity of rTgSRS2 ELISA were confirmed using a series of serum samples from T. gondii-experimentally infected mice. A total of 76 field samples from cats were examined by the developed ELISA. The rTgSRS2 ELISA showed a good diagnostic performance characterized by high concordance (88.16) and kappa value (0.76) with latex agglutination test (LAT). The sensitivity and specificity of the test were 92.68% and 82.86%, respectively. These results suggest that the ELISA based on rTgSRS2 could be a useful tool for serodiagnosis of T. gondii infection in cats.

Babesia, Theileria, and Anaplasma are important causative agents of tick-borne diseases that severely affect sheep. However, there is paucity in the occurrence genetic diversity of the infections of tick-borne diseases in sheep in border regions, northwestern China. In this study, nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. The most common dual infection was with A. ovis and B. motasi-like while the most frequent triple coinfection was A. ovis, B. motasi-like, and T. uilenbergi with coinfection rates of 17.0% (55/323) and 5.0% (16/323), respectively. Sequencing analysis indicated that A. ovis MSP4, A. phagocytophilum epank1, A. bovis 16S rRNA, B. motasi-like rap1-b, B. motasi-like Xinjiang rap1-a, T. luwenshuni 18S rRNA, and T. uilenbergi 18S rRNA from border regions, northwestern China, showed 99-100% identity with documented isolates from other countries. To the best of the authors' knowledge, this is the first report of T. uilenbergi and T. luwenshuni infections of sheep in border regions, northwestern China. Furthermore, these findings provide important data for understanding the distribution of Babesia, Theileria, and Anaplasma in sheep between border countries and China.

Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

Tick-borne diseases are of global economic importance, especially due to the costs associated with disease treatment and productivity losses in livestock. In this study, 244 livestock animals (cattle N = 92, buffaloes N = 86 and sheep N = 66) from Menoufia, Egypt were tested for Anaplasma, Ehrlichia and Babesia species using PCR. Results revealed detection of A. ovis (9.1%) in sheep while Anaplasma spp. (14.1%), A. marginale (15.2%), B. bigemina (6.5%) and B. bovis (5.4%) in cattle. On the other hand, Anaplasma spp. (1.2%), A. marginale (1.2%) and B. bovis (1.2%), were detected in buffaloes. Significantly higher detection rates were observed in cattle for Anaplasma spp. (P = .020), A. marginale (P = .001) and B. bigemina (P = .022) than in buffaloes. Sequence analysis of Anaplasma spp. isolates from cattle, revealed A. platys-like strains. Phylogenetic analyses of the A. platys-like isolates revealed variation among the strains infecting cattle. The A. marginale buffalo isolate, on the other hand, showed some level of divergence from the cattle isolates. This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt at large. The results of the current study provide valuable information on the epidemiology and genetic characteristics of tick-borne pathogens infecting livestock in Egypt.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. In Tanzania, detailed studies on tick-borne pathogens in cattle using sensitive molecular detection methods are scarce. In this study, we investigated the occurrence of Theileria spp., Babesia spp., Anaplasma spp. and Ehrlichia spp. in 245 blood samples collected from cattle on Pemba Island, Tanzania. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening revealed overall infection rates of 62.4% for Theileria spp., 17.6% for Babesia bigemina, 15.9% for Anaplasma marginale, 7.4% for Ehrlichia ruminantium and 4.5% for Babesia bovis. Further analysis using sequences of Theileria spp. 18S rRNA revealed infection of cattle with Theileria mutans (68.6%), Theileria taurotragi (48.4%), Theileria parva (41.2%), and Theileria ovis (1.9%). Co-infections of cattle, with up to six tick-borne pathogens, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 genes are conserved among cattle blood samples in Pemba, with 99.3%–100%, 99.6%–100% and 100% sequence identity values, respectively. In contrast, the B. bigemina RAP-1a and B. bovis SBP-2 gene sequences were relatively diverse with 99.5%–99.9% and 66.4%–98.7% sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina RAP-1 and B. bovis SBP-2 gene sequences showed significant differences in the genotypes, as they appeared in separate clades. This study provides important data for understanding the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoni in vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC50) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.

In Uganda, ticks and tick-borne diseases (TBDs) pose a big challenge to farmers. They reduce cattle productivity and cause severe economic damage. Several studies have documented the prevalence of tick-borne pathogens in cattle; however, their genetic characteristics and the role of wildlife-livestock interaction in the epidemiology of the TBDs are not well documented. This study assessed the prevalence and genetic diversity of various tick-borne pathogens (TBPs) as well as the risk factors associated with the occurrence of TBPs in blood samples of 208 randomly selected cattle from 16 farms located around Queen Elizabeth National Park (QENP) in Kasese District in western Uganda. Farming practices, disease challenges, and the level of wildlife-livestock interactions were assessed by a questionnaire survey amongst farm owners. Polymerase chain reaction (PCR) assays revealed that 62.9% (131/208) cattle samples were positive for one or more pathogens. Using specific PCR assays, we detected Theileria spp., Theileria parva, Anaplasma marginale, Anaplasma platys-like, and Babesia bigemina at 50.5%, 27.9%, 19.2%, 11.5% and 8.7%, respectively. We also confirmed the infection of samples by Theileria velifera and Theileria mutans after sequencing the Theileria spp. 18S rRNA gene. The risk factors associated with the occurrence of TBPs included communal grazing, herd size, age, and proximity to QENP. Phylogenetic analysis of the T. parva p104 gene showed a high identity to the previous isolates from Uganda and other East African countries and clustered closer to the buffalo (Syncerus caffer) isolates, suggesting a possible cross-species transmission. The sequences of A. marginale groEL and B. bigemina RAP-1a formed well-supported clades with high identities to the previous isolates identified from central and eastern Uganda. The isolates obtained from A. phagocytophilum 16S rRNA gene sequences showed relationship with A. platys-like, Anaplasma sp., uncultured Anaplasma species and A. phagocytophilum isolates from Africa, Asia, Europe, and the USA. The findings of the present study showed that TBDs are still a burden to farmers and that management practices in this area may increase the transmission of pathogens between livestock and wildlife.

Common vector-borne diseases of horses include equine piroplasmosis (EP) caused by Babesia caballi and Theileria equi, and equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum. Equine piroplasmosis leads to severe health issues in horses and restrictions on the movement of horses internationally. Anaplasma phagocytophilum causes an acute febrile illness in horses and is also of zoonotic importance. In the present study, blood samples were collected from 152 Turkish racehorses from three different provinces (Izmir, Gaziantep, and Konya) of Turkey to investigate the prevalence of EP and EGA. Standard and nested polymerase chain reactions were performed to identify equine piroplasms and A. phagocytophilum, respectively. PCR primers targeting Babesia spp. 18S rRNA, B. caballi BC48, T. equi EMA-1, and A. phagocytophilum 16S rRNA genes were used for molecular diagnosis. Following the cloning and subsequent sequencing of PCR-positive samples, a total of 15 (9.9%) horses were found to be infected with at least one pathogen. Theileria equi and A. phagocytophilum were found in 3.3% (5/152) and 6.6% (10/152) of the samples, respectively. Although B. caballi specimens were not detected in any of the samples, a positive signal was detected for the Babesia genus-specific 18S rRNA PCR. Subsequent sequencing of this signal revealed 100% identity to Babesia ovis. This is the first detection of B. ovis DNA in racehorses in Turkey to the best of our knowledge. Additionally, this study also reports the first molecular identification of A. phagocytophilum in Turkish racehorses. Based on this report, it is recommended that future epidemiological studies on horses also take B. ovis, a parasite usually found in sheep, into consideration and that further detailed studies be conducted to unravel the transmission pathways and potential clinical effects of B. ovis in horses.

Tick-borne diseases (TBDs) are serious constraints to livestock production in Tanzania and other tropical and subtropical countries and impact the livelihoods of resource-poor farming communities in the region. In Tanzania, detailed studies on tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to investigate the occurrence and species composition of bovine TBPs in cattle kept in Zanzibar Island. A total of 236 blood samples were randomly collected in cattle population in June and July 2019. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening of all 236 samples revealed that 64.5% of animals were infected by TBPs, including Theileria parva (34.3%), T. mutans (38.1%), T. taurotragi (30.9%), Anaplasma marginale (10.2%), Babesia bigemina (5.1%), T. velifera (3.4%) and B. bovis (2.1%). Overall a total of 86 animals (36.4%) were co-infected with up to five pathogens including T. parva, T. mutans, T. taurotragi, A. marginale and B. bigemina. The pathogens mostly involved in the co-infection were T. parva, T. taurotragi and T. mutans. Sequence analysis indicated that T. parva p104 and B. bigemina RAP1a genes are diverse among the sampled animals in Zanzibar Island, with 99.64%–100% and 99.51%–100% nucleotide sequence identity value respectively. In contrast, the A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 genes are conserved, with 100%, 99.05%-100% and 99.66%-100% nucleotide sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104 and B. bigemina RAP1a gene sequences showed significant differences of genotypes, as they appear in different clades. Meanwhile, A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 gene sequences appear in the same clade with other sequences extracted from the NCBI GenBank. The epidemiological findings revealed in this study will provide important information on tick-borne diseases in Tanzania and will be used as scientific basis for planning future control strategies.

Due to the specific plateau climate, a variety of unique animals live in the Qinghai-Tibetan Plateau Area (QTPA) including yaks (Bos grunniens), Tibetan sheep (Ovis aries) and Bactrian camels (Camelus bactrianus). However, information on tick-borne diseases (TBDs) in the QTPA and on the molecular characteristics of tick-borne pathogens (TBPs) in the area is limited. Therefore, the aim of this study was to investigate Anaplasma spp., Babesia spp., Theileria spp., Borrelia burgdorferi sensu lato and Rickettsia spp. infecting yaks, Tibetan sheep and camels in this area. A total of 276 animals were screened. Overall, 84.5% (164/194) of yaks, 58% (23/40) of Tibetan sheep, and 38% (16/42) of camels tested positive for at least one pathogen. Theileria spp., Anaplasma ovis and spotted fever group (SFG) Rickettsia spp. were detected as TBPs in the current study with overall infection rates of 10.9% (30/276), 8.3% (23/276) and 62.9% (171/276), respectively. Further study revealed that 1.5% (3/194) of the yaks were infected with Theileria sp. OT3, 1.5% (3/194) with T. luwenshuni, 6.2% (12/194) with T. uilenbergi, 1.1% (2/194) with T. ovis and 82% (159/194) with SFG Rickettsia spp. It was also shown that 58% (23/40) of the Tibetan sheep were infected with A. ovis and 15% (6/40) with T. ovis. Among the camels, 10% (4/42) were infected with T. equi, while 29% (12/42) were positive for Rickettsia spp. Sequence analysis revealed that the Rickettsia spp. infecting yaks and camels were Rickettsia raoultii and Rickettsia slovaca. To the best of our knowledge, this study reports the first detection and characterization of these pathogens in yaks, Tibetan sheep and camels in the country, except for T. luwenshuni infections in yaks.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 μM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

Q fever, spotted fever rickettsioses and equine piroplasmosis, are some of the most serious equine tick-borne diseases caused by Coxiella burnetii, Rickettsia spp., Babesia caballi and/or Theileria equi. This study surveyed and molecularly characterized these pathogens infecting horses in ten ranches from XUAR, China using molecular technology. Among 200 horse blood samples, 163 (81.5%) were infected with at least one of the pathogens. Rickettsia spp. was the most prevalent pathogen (n = 114, 57.0%), followed by C. burnetii (n = 79, 39.5%), T. equi (n = 79, 39.5%) and B. caballi (n = 49, 24.5%). Co-infections were observed in 61.3% of positive samples in this study. Statistically significant differences were observed between the sampling regions for C. burnetii, B. caballi and T. equi, and also in different age group for C. burnetii and T. equi. The genotype analysis indicated that C. burnetii htpB, Rickettsia spp. ompA, B. caballi rap-1, B. caballi 18S rRNA, T. equi EMA-1 and T. equi 18S rRNA gene sequences from horses in XUAR were variable. To the best of our knowledge, this study is the first report of C. burnetii and Rickettsia spp. infection and co-infected with piroplasma in horses in China. Overall, this study revealed the high infection rate of the pathogens in horses in XUAR, China. The current findings are expected to provide a basis for better tick-borne disease control in the region.