A multipronged next-generation sequencing metabarcoding approach unearths hyperdiverse and abundant dog pathogen communities in Cambodia.

Recent surveys in Southeast Asia, including Cambodia, have identified canine vector-borne pathogens (VBPs), including those with zoonotic potential, as highly prevalent. The lack of veterinary care alongside the close association semidomesticated dogs have with humans in the region exacerbates these zoonotic risks. Nonetheless, the number of studies investigating such pathogens and the threats they pose to dog and human health is limited. Here, we utilize a next-generation sequencing (NGS)-based metabarcoding protocol to conduct an assumption-free characterization of the bacterial, apicomplexan, and kinetoplastid blood-borne pathogens of free-roaming dogs from across Cambodia. From 467 dogs at five field sites, 62% were infected with one of eight confirmed pathogens, comprising Anaplasma platys (32%), Ehrlichia canis (20%), Hepatozoon canis (18%), Babesia vogeli (14%), Mycoplasma haemocanis (13%), the zoonotic pathogen Bartonella clarridgeiae (3%), Candidatus Mycoplasma haematoparvum (0.2%), and Trypanosoma evansi (0.2%). Coinfections of between two and four VBPs were common with 28% of dogs found to have a mixed infection. Moreover, DNA from putatively infectious agents belonging to the bacterial family and genera Coxiella, Mycobacterium, Neisseria, Rickettsiaceae, Treponema, and two uncharacterized Mycoplasma species were identified, in addition to protozoan genera Colpodella, Parabodo, and Bodo. Using a multiple logistic regression model, the presence of ectoparasites, abnormal mucous membranes, anemia, and total protein were found as predictors of canine VBP exposure. This study represents the first time an NGS metabarcoding technique has been used to holistically detect the bacterial and protozoan hemoparasites communities of dogs through an in-depth survey, highlighting the power of such methods to unearth a wide spectrum of pathogenic organisms in an unbiased manner.

The effects of trans-chalcone and chalcone 4 hydrate on the growth of Babesia and Theileria.

BACKGROUND: Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone 4 hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. METHODOLOGY/PRINCIPAL FINDINGS: The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on four Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin-Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC-DA and TC-CF were more potent against B. microti infection in mice than their monotherapies. CONCLUSIONS/SIGNIFICANCE: In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies.

Toward the discovery of inhibitors of babesipain-1, a Babesia bigemina cysteine protease: in vitro evaluation, homology modeling and molecular docking studies.

Babesia bigemina is a protozoan parasite that causes babesiosis, a disease with a world-wide distribution in mammals, principally affecting cattle and man. The unveiling of the genome of B. bigemina is a project in active progress that has already revealed a number of new targets with potential interest for the design of anti-babesiosis drugs. In this context, babesipain-1 has been identified as a proteolytically active enzyme whose three-dimensional structure has not been resolved yet, but which is known to be inhibited by cysteine proteases inhibitors such as E64, ALLN, leupeptin, and vinyl sulfones. In this work, we introduce (1) a homology model of babesipain-1; (2) a comparison between babesipain-1 and falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum; (3) in vitro data for babesipain-1 inhibition by HEDICINs and HECINs, previously reported as modest inhibitors of falcipain-2; and (4) the docked binding conformations of HEDICINs and HECINs in the model of babesipain-1. HEDICINs presented similar preferred binding conformations for both babesipain-1 and falcipain-2. However, in vitro bioassay shows that HEDICINs and HECINs are better inhibitors of babesipain-1 than of falcipain-2, which could be explained by observed differences between the active pockets of these proteins in silico. Results presented herein provide a valuable contribution to future computer-aided molecular design of new babesipain-1 inhibitors.

Molecular detection and characterization of potentially new Babesia and Theileria species/variants in wild felids from Kenya.

Piroplasms frequently infect domestic and wild carnivores. At present, there is limited information on the occurrence and molecular identity of these tick-borne parasites in wild felids in Kenya. In 2009, a pair of captive lions (Panthare leo) was diagnosed with suspected babesiosis and mineral deficiency at an animal orphanage on the outskirts of Nairobi, Kenya. Blood smears indicated presences of haemoparasites in the erythrocytes, however, no further investigations were conducted to identify the infecting agent. The animals recovered completely following diet supplementation and treatment with anti-parasite drug. In this report, we extracted and detected parasite DNA from the two lions and seven other asymptomatic feline samples; two leopards (Panthera pardus) and five cheetahs (Acinonyx jubatus). Reverse line blot with probes specific for Babesia spp. of felines indicated the presence of new Babesia species or genotypes in the lions and leopards, and unknown Theileria sp. in the cheetahs. Phylogenetic analyses using partial sequences of 18S ribosomal RNA (18S rRNA) gene showed that the parasite infecting the lions belong to the Babesia canis complex, and the parasite variant detected in the leopards clusters in a clade bearing other Babesia spp. reported in wild felids from Africa. The cheetah isolates falls in the Theileria sensu stricto group. Our findings indicate the occurrence of potentially new species or genotypes of piroplams in all three feline species.

Evaluation of efficacy of bruceine A, a natural quassinoid compound extracted from a medicinal plant, Brucea javanica, for canine babesiosis.

Bruceine A, a natural quassinoid compound extracted from the dried fruits of Brucea javanica (L.) Merr., was evaluated for its antibabesial activity in vitro and in vivo. Bruceine A inhibited the in vitro growth of Babesia gibsoni in canine erythrocytes at lower concentration compared with the standard antibabesial drug diminazene aceturate and killed the parasites within 24 hr at a concentration of 25 nM. Oral administration of bruceine A at a dosage of 6.4 mg/kg/day for 5 days resulted in no clinical findings in a dog with normal ranges of hematological and biochemical values in the blood. Three dogs were infected with B. gibsoni and two of them were treated with bruceine A at a dosage of 6.4 mg/kg/day for 6 days from day 5 post-infection. An untreated dog developed typical acute babesiosis symptoms including severe anemia, high fever, and complete loss of appetite and movement. However, the two bruceine A-treated dogs maintained their healthy conditions throughout the experimental period of 4 weeks although complete elimination of parasites from the peripheral blood was not achieved and decreases in the packed cell volume and the erythrocyte and platelet counts were observed. Since natural quassinoid compounds have been used as traditional medicines for the treatment of various ailments including cancer and malaria, the present results suggest that bruceine A or other related compounds are potential candidates for the treatment of canine babesiosis.