Molecular identification and characterization of piroplasm species in Hokkaido sika deer (Cervus nippon yesoensis), Japan

Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the generaIxodesandHaemaphysalis. In total 163 samples (31.4%) were positive forBabesiaandTheileriaspp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes ofTheileriasp.Thrivae (27.0% of ticks; 140/519),Theileria capreoli(10.6%; 55/519),Babesia divergens-like (1.7%; 9/519),Babesiasp. (Bab-SD) (0.6%; 3/519),Babesia microtiU.S. (0.4%; 2/519), andB. microtiHobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens

The Prevalence and Molecular Biology of Staphylococcus aureus Isolated from Healthy and Diseased Equine Eyes in Egypt

This work aimed to characterize S. aureus isolates from the eyes of healthy and clinically affected equines in the Kafrelsheikh Governorate, Egypt. A total of 110 animals were examined for the presence of S. aureus, which was isolated from 33 animals with ophthalmic lesions and 77 healthy animals. We also investigated the antimicrobial resistance profile, oxacillin resistance mechanism, and the major virulence factors implicated in many studies of the ocular pathology of pathogenic S. aureus. The association between S. aureus eye infections and potential risk factors was also investigated. The frequency of S. aureus isolates from clinically affected equine eyes was significantly higher than in clinically healthy equids. A significant association was found between the frequency of S. aureus isolation from clinically affected equine eyes and risk factors including age and season but not with sex or breed factors. Antimicrobial resistance to common antibiotics used to treat equine eyes was also tested. Overall, the isolates showed the highest sensitivity to sulfamethoxazole (100%) and the highest resistance to cephalosporin (90.67%) and oxacillin (90.48%). PCR was used to demonstrate that mecA was present in 100% of oxacillin- and β-lactam-resistant S. aureus strains. The virulence factor genes Spa (x region), nuc, and hlg were identified in 62.5%, 100%, and 56%, of isolates, respectively, from clinically affected equines eyes. The severity of the eye lesions increased in the presence of γ-toxin-positive S. aureus. The phylogenetic tree of the Spa (x region) gene indicated a relationship with human reference strains isolated from Egypt as well as isolates from equines in Iran and Japan. This study provides insight into the prevalence, potential risk factors, clinical pictures, zoonotic potential, antimicrobial resistance, and β-lactam resistance mechanism of S. aureus strains that cause eye infection in equines from Egypt

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against <i>Babesia</i> and <i>Theileria</i> Parasites

<div><p>A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against <i>Babesia</i> parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in <i>in vitro</i> cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine <i>Babesia</i> parasites and 5% for equine <i>Babesia</i> and <i>Theileria</i> parasites. The IC₅₀ values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC₅₀ values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for <i>in vitro</i> large-scale drug screening against <i>Babesia</i> and <i>Theileria</i> parasites that infect cattle and horses.</p></div

IC₅₀ values of the diminazene aceturate drug evaluated for <i>B</i>. <i>bovis</i> and <i>T</i>. <i>equi</i> with and without daily replacement of the medium using the fluorescence-based method.

<p>^a IC₅₀ values for diminazene aceturate were determined on the fourth day of <i>in vitro</i> culture using the fluorescence-based method with 2.5% and 5% HCTs for <i>B</i>. <i>bovis</i> and <i>T</i>. <i>equi</i>, respectively, in three separate experiments. Each drug concentration was made in triplicate in each experiment, and the final obtained IC₅₀s were the mean of three obtained values from separate experiments.</p><p>^b The differences between IC₅₀ values determined using the fluorescence-based method with and without daily replacement of the medium were not statistically significant.</p><p>IC₅₀ values of the diminazene aceturate drug evaluated for <i>B</i>. <i>bovis</i> and <i>T</i>. <i>equi</i> with and without daily replacement of the medium using the fluorescence-based method.</p

Growth inhibition of <i>B</i>. <i>bovis</i>, <i>B</i>. <i>bigemina</i>, <i>T</i>. <i>equi</i>, and <i>B</i>. <i>caballi</i> by diminazene aceturate on the fourth day estimated by the fluorescence-based method (black) and microscopy (gray).

<p>(A) Growth inhibition of <i>B</i>. <i>bovis</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). (B) Growth inhibition of <i>B</i>. <i>bigemina</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). (C) Growth inhibition of <i>T</i>. <i>equi</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). (D) Growth inhibition of <i>B</i>. <i>caballi</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). Each value is presented as the mean of three triplicate wells ± SD.</p

Growth inhibition of <i>B</i>. <i>bovis</i> and <i>T</i>. <i>equi</i> by diminazene aceturate estimated by the fluorescence-based method without daily replacement of the medium (black) and with daily replacement of the medium (gray).

<p>(A) Growth inhibition of <i>B</i>. <i>bovis</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). (B) Growth inhibition of <i>T</i>. <i>equi</i> (y-axis) and log concentration of diminazene aceturate (nM) (x-axis). Each value is presented as the mean of three triplicate wells ± SD after subtraction of the background fluorescence for non-parasitized RBCs.</p