Bioinformatics approach for structure modeling, vaccine design, and molecular docking of Brucella candidate proteins BvrR, OMP25, and OMP31.

Brucellosis is a zoonotic disease with significant economic and healthcare costs. Despite the eradication efforts, the disease persists. Vaccines prevent disease in animals while antibiotics cure humans with limitations. This study aims to design vaccines and drugs for brucellosis in animals and humans, using protein modeling, epitope prediction, and molecular docking of the target proteins (BvrR, OMP25, and OMP31). Tertiary structure models of three target proteins were constructed and assessed using RMSD, TM-score, C-score, Z-score, and ERRAT. The best models selected from AlphaFold and I-TASSER due to their superior performance according to CASP 12 - CASP 15 were chosen for further analysis. The motif analysis of best models using MotifFinder revealed two, five, and five protein binding motifs, however, the Motif Scan identified seven, six, and eight Post-Translational Modification sites (PTMs) in the BvrR, OMP25, and OMP31 proteins, respectively. Dominant B cell epitopes were predicted at (44-63, 85-93, 126-137, 193-205, and 208-237), (26-46, 52-71, 98-114, 142-155, and 183-200), and (29-45, 58-82, 119-142, 177-198, and 222-251) for the three target proteins. Additionally, cytotoxic T lymphocyte epitopes were detected at (173-181, 189-197, and 202-210), (61-69, 91-99, 159-167, and 181-189), and (3-11, 24-32, 167-175, and 216-224), while T helper lymphocyte epitopes were displayed at (39-53, 57-65, 150-158, 163-171), (79-87, 95-108, 115-123, 128-142, and 189-197), and (39-47, 109-123, 216-224, and 245-253), for the respective target protein. Furthermore, structure-based virtual screening of the ZINC and DrugBank databases using the docking MOE program was followed by ADMET analysis. The best five compounds of the ZINC database revealed docking scores ranged from (- 16.8744 to - 15.1922), (- 16.0424 to - 14.1645), and (- 14.7566 to - 13.3222) for the BvrR, OMP25, and OMP31, respectively. These compounds had good ADMET parameters and no cytotoxicity, while DrugBank compounds didn't meet Lipinski's rule criteria. Therefore, the five selected compounds from the ZINC20 databases may fulfill the pharmacokinetics and could be considered lead molecules for potentially inhibiting Brucella's proteins.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

Recent progress in the genotyping of bovine tuberculosis and its rapid diagnosis via nanoparticle-based electrochemical biosensors.

Bovine tuberculosis (bTB) is considered a worldwide infectious zoonotic disease. Mycobacterium bovis causes bTB disease. It is one of the Mycobacterium tuberculosis complex (MTBC) members. MTBC is a clonal complex of close relatives with approximately 99.95% similarity. M. bovis is a spillover pathogen that can transmit from animals to humans and rarely from humans to animals with contact. Genotyping techniques are important to discriminate and differentiate between MTBC species. Spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) are widely used but they have some limitations. As an alternative, whole genome sequencing approaches have been utilized due to their high-resolution power. They are employed in typing M. bovis and explain the evolutionary and phylogenetic relationships between isolates. The control of bTB disease has attracted a large amount of attention. Rapid and proper diagnosis is necessary for monitoring the disease as an initial step for its control and treatment. Nanotechnology has a potential impact on the rapid diagnosis and treatment of bTB through the use of nanocarrier and metal nanoparticles (NPs). Special attention has been paid to voltammetric and impedimetric electrochemical strategies as facile, sensitive, and selective methods for the efficient detection of tuberculosis. The efficacy of these sensors is enhanced in the presence of NPs, which act as recognition and/or redox probes. Gold, silver, copper, cobalt, graphene, and magnetic NPs, as well as polypyrrole nanowires and multiwalled carbon nanotubes have been employed for detecting tuberculosis. Overall, NP-based electrochemical sensors represent a promising tool for the diagnosis of bTB.

Brucellae as resilient intracellular pathogens: epidemiology, host-pathogen interaction, recent genomics and proteomics approaches, and future perspectives.

Brucellosis is considered one of the most hazardous zoonotic diseases all over the world. It causes formidable economic losses in developed and developing countries. Despite the significant attempts to get rid of Brucella pathogens in many parts of the world, the disease continues to spread widely. Recently, many attempts proved to be effective for the prevention and control of highly contagious bovine brucellosis, which could be followed by others to achieve a prosperous future without rampant Brucella pathogens. In this study, the updated view for worldwide Brucella distribution, possible predisposing factors for emerging Brucella pathogens, immune response and different types of Brucella vaccines, genomics and proteomics approaches incorporated recently in the field of brucellosis, and future perspectives for prevention and control of bovine brucellosis have been discussed comprehensively. So, the current study will be used as a guide for researchers in planning their future work, which will pave the way for a new world without these highly contagious pathogens that have been infecting and threatening the health of humans and terrestrial animals.

Evaluation and Haematological Profile of an Inactivated RVF Vaccine Locally Produced in Egypt.

<b>Background and Objective:</b> The RVF virus cause diseases in newborn puppies, kittens, sheep, goats, cattle, camels, buffaloes and also humans. The RVF disease was first detected among livestock by veterinary officers. The disease causes abortions in animals. The goal of this study was to evaluate the immune response and the haematological profile associated with inactivated RFV vaccine locally produced in Egypt in young puppies and sheep. <b>Materials and Methods:</b> Through vaccination, both young puppies and sheep with local produced inactivated RVF vaccine with 2 doses with 2 weeks interval and evaluate the immune response by SNT and ELISA as well as haematological parameters at 0, 7, 14, 21 and 28 days post-vaccination. The variance between vaccinated groups and also non-vaccinated groups were compared by using a one-way Analysis of Variance (ANOVA). <b>Results:</b> The findings showed that young puppies had a strong response to antibodies after two doses of the RVF vaccine within the 2 week interval. The neutralization indices (NI) values in young puppies at different periods after RVF vaccination reported the value of 1.08±0.03, 1.23±0.04, 1.30±0.03 and 1.45±0.02 after 7, 14, 21 and 28 days post-vaccination, respectively. Parallel to this the ELISA OP values were 0.30±0.00, 0.39±0.03, 0.52±0.05 and 0.75±0.02 after 7, 14, 21 and 28 days post-vaccination, respectively. Nearly similar immune response was noticed in sheep with NI values of 1.15±0.02, 1.27±0.02, 1.42±0.05 and 1.55±0.03 at 7, 14, 21 and 28 days post-vaccination, respectively. In the same site the ELISA OP values were 0.34±0.00, 0.47±0.01, 0.68±0.00, 0.77±0.00. After 7, 14, 21 and 28 days post-vaccination respectively that are also similar to that in puppies. The haematological profile reported a significant decrease after the 1st week followed by a transient increase after booster dose at 2nd week except for the monocytes that increase after 1st week then decreases after 2nd week post-vaccination. <b>Conclusion:</b> Young puppies are similar to sheep in developing antibodies after vaccination with the RFV vaccine with no statistically significant effect within different batches. In addition, ELISA can replace the SNT for evaluation of the immune response. Young puppies are quite equal to sheep for the illustration of neutralizing antibodies for RFV vaccine. Sero-negative puppies can be easily obtained because dogs are not included in the vaccination program of RVF and so they can be used as a good model to determine the efficacy of the RVF vaccine.

Spatiotemporal analysis of highly pathogenic avian influenza (H5N1) outbreaks in poultry in Egypt (2006 to 2017).

BACKGROUND: In Egypt, the highly pathogenic avian influenza (HPAI) subtype H5N1 is endemic and possesses a severe impact on the poultry. To provide a better understanding of the distributional characteristics of HPAI H5N1 outbreaks in Egypt, this study aimed to explore the spatiotemporal pattern and identify clusters of HPAI H5N1 outbreaks in Egypt from 2006 to 2017. RESULTS: The Epidemic curve (EC) was constructed through time series analysis; in which six epidemic waves (EWs) were revealed. Outbreaks mainly started in winter peaked in March and ended in summer. However, newly emerged thermostable clades (2.2.1.1 and 2.2.1.2) during the 4th EW enabled the virus to survive and cause infection in warmer months with a clear alteration in the seasonality of the epidemic cycle in the 5th EW. The endemic situation became more complicated by the emergence of new serotypes. As a result, the EC ended up without any specific pattern since the 6th EW to now. The spatial analysis showed that the highest outbreak density was recorded in the Nile Delta considering it as the 'Hot spot' region. By the 6th EW, the outbreak extended to include the Nile valley. From spatiotemporal cluster epidemics, clustering in the Delta was a common feature in all EWs with primary clusters consistently detected in the hot-spot region, but the location and size varied with each EW. The highest Relative Risk (RR) regions in an EW were noticed to contain the primary clusters of the next EW and were found to include stopover sites for migratory wild birds. They were in Fayoum, Dakahlia, Qalyobiya, Sharkia, Kafr_Elsheikh, Giza, Behera, Menia, and BeniSuef governorates. Transmission of HPAI H5N1 occurred from one location to another directly resulted in a series of outbreaks forming neighboring secondary clusters. The absence of geographical borders between the governorates in addition to non-restricted movements of poultry and low vaccination and surveillance coverage contributed to the wider spread of infection all over Egypt and to look like one epidemiological unit. CONCLUSION: Our findings can help in better understanding of the characteristics of HPAI H5N1 outbreaks and the distribution of outbreak risk, which can be used for effective disease control strategies.

Clinical, histopathological, and molecular characterization of Mycoplasma species in sheep and goats in Egypt.

BACKGROUND AND AIM: Mycoplasma infection in small ruminants is a serious problem in sheep and goat herds around the world. It is responsible for high economic losses and decreased animal productivity. This study aimed to highlight the clinical, histopathological, minimum inhibitory concentration (MIC), and molecular characterization of Mycoplasma species in sheep and goats in Menoufiya Governorate, Egypt. MATERIALS AND METHODS: A total of 234 samples were collected; 104 samples were collected from pneumonic lung tissues from the abattoir, in addition, 10 and 20 samples collected from apparently and diseased sheep, respectively, and 40 and 60 samples were collected from apparently and diseased goats, respectively, which were subjected to isolation onto pleuropneumonia-like organism medium. Polymerase chain reaction (PCR), histopathological examination, and determination of the MIC were also performed. RESULTS: Of 104 samples of lung tissues showing pneumonic lesions, 56 (53.84%) were positive for Mycoplasma isolation. The positive isolation of Mycoplasma from 10 and 20 samples from apparently and diseased sheep was 30% and 40%, respectively as well as the positive isolation of Mycoplasma was 17% and 56.66% out of 40 and 60 apparently healthy and diseased field goat's cases, respectively. All the diseased sheep and goats showed respiratory manifestations, including cough, bilateral nasal discharge, conjunctivitis, and systemic reaction. Evaluation of the MIC for Mycoplasma ovipneumoniae revealed that lincospectin and tylosin were the most effective antibiotics at 2.5 mg/mL. Histopathological examination of affected lung tissue showed extensive hemorrhagic pneumonia with extensive alveolar hemorrhage. The PCR technique proved to be a rapid, specific, and sensitive method for the detection of M. ovipneumoniae and Mycoplasma arginini at 390 and 326 bp, respectively. CONCLUSION: M. ovipneumoniae and M. arginini were the most prevalent species associated with respiratory infections in sheep and goats in the study area. Further studies are needed to investigate the role of these species in dissemination of the disease within herds of small ruminants.

Hepatic biomarkers and coprology as indicators of clinical bovine fasciolosis in Chad.

BACKGROUND AND AIM: Fasciolosis is a cosmopolitan parasitic disease of food-producing animals and is typically caused by digenetic trematodes, Fasciola gigantica and Fasciola hepatica. It has a direct negative impact on the liver and consequently affects liver metabolism. It has indirect effects, including lowered milk production and effects on quality and general health conditions leading to extensive economic losses. This study aimed to focus on the link between clinical fasciolosis and some biochemical analysis of the hepatic profile of cattle in Chad. MATERIALS AND METHODS: This study was initiated in response to emerging complaints from dairy cattle owners detecting a bitter milk cream and butter taste. Furthermore, those animals had shown poor health conditions by presenting with diarrhea. Preliminary surveillance for possible causes was performed, including fecal and serum biochemical analyses and clinical observation to diagnose the possible disease. RESULTS: The results obtained, including the finding of parasite stages during the coprological examination, confirmed the role of fasciolosis. The independent sample t-test indicated highly significantly altered values of all biochemical liver indicators in the infected animals. All animals were treated with two doses of rafoxanide (3 mg/kg b.w.) S/C, at 21 days intervals, with vitamin supplements, mineral mixtures, and food additives. Surprisingly, the main complaint was restored after treatment. This is another evident clue of fasciolosis. To the best of our knowledge, this is the first recent study that diagnosed fasciolosis in Chad. CONCLUSION: This study emphasized the importance of fasciolosis, its negative impact on milk taste, and the necessity for veterinary advice regarding routine examination and prophylactic measures, especially before autumn, to minimize economic losses. However, regardless of the small sample size, this study could serve as a cornerstone for future studies on evaluating the accurate epidemiological status of fasciolosis in Chad. This study reported a close association between the alteration of liver enzymes and total protein levels in fasciolosis and the bitter milk cream taste, which could be used as a diagnostic tool for fasciolosis.

Phylogenetic study of Theileria ovis and Theileria lestoquardi in sheep from Egypt: Molecular evidence and genetic characterization.

BACKGROUND AND AIM: Ovine theileriosis caused by Theileria ovis and Theileria lestoquardi is an important infectious disease affecting small ruminants in regions of the tropic and subtropic zones. There is limited studies about ovine theileriosis in Egypt; so the present study aims to assess the occurrence of ovine theileriosis in Egypt at the molecular level. MATERIALS AND METHODS: Blood samples were collected from 115 randomly selected sheep, which were apparently healthy; the ages of the sampled sheep ranged from 1 to 5 years old, from a local breed (barkae and balade), and showed no symptoms indicating infection with Theileria spp. The study was conducted in three governorates representing Lower Egypt (Menoufia and Beheira) and Upper Egypt (El-Wady El-Geded). All blood samples were subjected to polymerase chain reaction (PCR) and semi-nested PCR to target Theileria spp. 18S rRNA genes. Positive samples were sequenced, and these sequences were analyzed using nucleotidebasic local alignment search tool (BLAST). RESULTS: Six animals (5.22%) were PCR-positive carriers for ovine theileriosis. Nucleotide BLAST and phylogenetic analyses of the six obtained sequences showed that T. ovis was present in five animals (4.37%) in Menoufia (n=2) and El-Wady El-Geded (n=3), whereas T. lestoquardi was detected in 1 animal (0.87%) in El-Wady El-Geded. CONCLUSION: This study is the first to provide molecular evidence, genetic characterization, and phylogenetic analysis of ovine Theileria spp. in Egypt. Specifically, T. lestoquardi and T. ovis carrier statuses of sheep were confirmed. These results highlight the importance of developing an effective control strategy against ovine theileriosis carriers that might develop and/or spread theileriosis.

Depression among Infertile Women in Gaza Strip: Symptom Severity and Predictors.

BACKGROUND: Mental disorders are expected for women suffering from infertility. Depression is a predictable consequence but requires more investigations and considerations. This study is aimed at determining the severity of depression symptoms and its predictors among infertile women in the Gaza Strip, Palestine. MATERIALS AND METHODS: A cross-sectional study was conducted. Three hundred eighty-five infertile women participated and were selected by convenience sampling. The participated women were selected from three large and major in vitro fertilization treatment centers (Al Helo, Al Basma, and Hindawi). A validated Arabic version of the Beck Depression Inventory-II was used. Univariate and multivariate logistic regression was applied to determine potential predictors for depression symptoms, and p ≤ .05 was statistically significant. RESULTS: The mean age of participated women was 29 ± 6.58 years, and the mean duration of marriage and infertility was 7.76 ± 5.31 and 5.43 ± 3.50 years, respectively. Half of the women had some form of depression symptoms (22.3%, 8.6%, and 10.6% showed to have mild depression, moderate, and severe depression symptoms, respectively). Predictors of depression symptoms were as follows: duration of marriage (Wald test: 10.493; CI95%: 0.248-0.774), at least one abortion (Wald test: 21.233; CI95%: 1.863-4.528), primary infertility (Wald test: 6.666; CI95%: 1.148-2.742), and husband cause of infertility (Wald test: 10.878; CI95%: 0.800-0.982). CONCLUSION: Infertility affects the various aspect of women's life. Psychological intervention including counselling, support, and therapy would be necessary to limit the consequences of infertility. Such interventions could be implemented in infertility treatment clinics.

Cardiac troponin I as a cardiac biomarker has prognostic and predictive value for poor survival in Egyptian buffalo calves with foot-and-mouth disease.

BACKGROUND AND AIM: Foot-and-mouth disease (FMD) causes huge economic losses in Egypt due to reductions in the production of red meat, milk, and milk by-products and can also lead to myocarditis in young animals. The aim of our study was to evaluate cardiac biomarkers, in particular cardiac troponin I (cTnI), and to reveal the relations of cardiac biomarkers with poor survival in FMD-infected Egyptian buffalo calves. MATERIALS AND METHODS: Forty-two Egyptian buffalo calves were included in this study. The calves were divided into 12 apparently healthy control calves and 30 calves clinically diagnosed with FMD during a disease outbreak in Menofia and Behera Governorates, Egypt. The diseased calves were divided, according to age, into 13 calves <3 months old and 17 calves between 3 and 6 months old. The animals were examined clinically and subjected to analysis of cardiac biomarkers. RESULTS: Biochemical analysis revealed significant elevations of cardiac biomarkers, especially creatine kinase myocardial band (CK-MB), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), cardiac troponin T (cTnT), and cardiac troponin I (cTnI) in FMD-infected calves in comparison with control calves. There was a significant association between cTnI and poor survival in infected calves. CONCLUSION: Cardiac biomarkers could be used as a rapid method for diagnosis of myocarditis induced by FMD in Egyptian buffalo calves. In addition, cTnI is a very sensitive and accurate tool for determining myocardial cell damage in the earlier stages of the disease and a good predictor of poor survival in calves.

Using phylogeographic approaches to analyse the dispersal history, velocity and direction of viral lineages - Application to rabies virus spread in Iran.

Recent years have seen the extensive use of phylogeographic approaches to unveil the dispersal history of virus epidemics. Spatially explicit reconstructions of viral spread represent valuable sources of lineage movement data that can be exploited to investigate the impact of underlying environmental layers on the dispersal of pathogens. Here, we performed phylogeographic inference and applied different post hoc approaches to analyse a new and comprehensive data set of viral genomes to elucidate the dispersal history and dynamics of rabies virus (RABV) in Iran, which have remained largely unknown. We first analysed the association between environmental factors and variations in dispersal velocity among lineages. Second, we present, test and apply a new approach to study the link between environmental conditions and the dispersal direction of lineages. The statistical performance (power of detection, false-positive rate) of this new method was assessed using simulations. We performed phylogeographic analyses of RABV genomes, allowing us to describe the large diversity of RABV in Iran and to confirm the cocirculation of several clades in the country. Overall, we estimate a relatively high lineage dispersal velocity, similar to previous estimates for dog rabies virus spread in northern Africa. Finally, we highlight a tendency for RABV lineages to spread in accessible areas associated with high human population density. Our analytical workflow illustrates how phylogeographic approaches can be used to investigate the impact of environmental factors on several aspects of viral dispersal dynamics.

Serum vitamin A and E, copper, zinc and selenium concentrations and their relationship with health outcomes in dromedary hospitalized camels (Camelus dromedarius).

The goals of this study were to measure serum vitamin A (retinol) and E (α-tocopherol) and trace elements concentrations (copper, zinc and selenium) during diseases condition and to determine their association with hematological parameters and immune status of hospitalized camels. A total of 95 dromedary camels [healthy (n=65); hospitalized camels (n=30)] were included in this study. Vitamin A and E concentrations were significantly lower in hospitalized camels than apparently healthy ones (P<0.05). Hospitalized camels had lower concentrations of zinc and selenium compared to healthy camels (P<0.05). Vitamin E, copper, zinc and selenium concentrations were positively correlated with phagocytic activity in hospitalized camels (P<0.05). The likelihood of deficiency of vitamin A and E, zinc and selenium concentrations were significant in female hospitalized camels than males and in young age hospitalized camels < 6 years old compared to old ones (P<0.05). Decreased vitamin A and E and trace elements concentrations were associated with hospitalized camels' phagocytic activity and index. The prevalence of low vitamin A and E, zinc and selenium concentrations were frequent in female hospitalized camels and hospitalized camels of age < 6 years old suggesting severe oxidative stress.

Correction: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules.

[This corrects the article DOI: 10.1371/journal.pone.0185372.].

Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules.

Human babesiosis is caused by the apicomplexan parasite Babesia microti, which is of major public health concern in the United States and elsewhere, resulting in malaise and fatigue, followed by a fever and hemolytic anemia. In this paper we focus on the characterization of a novel B. microti thrombospondin domain (TSP1)-containing protein (BmP53) from the new annotation of the B. microti genome (locus 'BmR1_04g09041'). This novel protein (BmP53) had a single TSP1 and a transmembrane domain, with a short cytoplasmic tail containing a sub-terminal glutamine residue, but no signal peptide and Von Willebrand factor type A domains (VWA), which are found in classical thrombospondin-related adhesive proteins (TRAP). Co-localization assays of BmP53 and Babesia microti secreted antigen 1 (BmSA1) suggested that BmP53 might be a non-secretory membranous protein. Molecular mimicry between the TSP1 domain from BmP53 and host platelets molecules was indicated through different measures of sequence homology, phylogenetic analysis, 3D structure and shared epitopes. Indeed, hamster isolated platelets cross-reacted with mouse anti-BmP53-TSP1. Molecular mimicry are used to help parasites to escape immune defenses, resulting in immune evasion or autoimmunity. Furthermore, specific host reactivity was also detected against the TSP1-free part of BmP53 in infected hamster sera. In conclusion, the TSP1 domain mimicry might help in studying the mechanisms of parasite-induced thrombocytopenia, with the TSP1-free truncate of the protein representing a potential safe candidate for future vaccine studies.

Animal level risk factors associated with Babesia and Theileria infections in cattle in Egypt.

In present study, blood samples were collected randomly from 439 cows at three main regions of Egypt (northern, central and southern). Molecular diagnosis of Babesia and Theileria infections by PCR amplification of DNA (gene) fragments, then cloning and sequencing of the positive samples were conducted. A questionnaire was created to imply the assumed risk factors and logistic regression statistical analysis was carried out to appraise the potential factors on the animal level. The results revealed that 49 (11.16%) and 45 (10.25%) cattle were infected with Babesia and Theileria parasites, respectively. B. bigemina (7.97%) and T. annulata (9.56%) were the most prevalent parasites. For Babesia sp., final multivariate logistic regression analysis showed a significant association between the infection and irregular use of antiprotozoal drugs (P = 0.003; OR: 0.28; 95% CI: 0.12-0.65), management practice (P = 0.029; OR: 6.66; 95% CI: 1.21-36.59) and ecology area (P = 0.006; OR: 5.62; 95% CI: 1.63-19.31). However, for Theileria sp. infection, animal breed (P = 0.003; OR: 0.44; 95% CI: .45-1.00) and irregular use of antiprotozoal drugs (P<0.001; OR: 4.22; 95% CI: 2.62-5.60) were the potential risk factors. The results of the present study declare the prevalent bovine Babesia and Theileria sp. in Egypt based on molecular description. An impression on the potential risk factors associated with infections was obtained. Recognition of the potential risk factors associated with tick borne disease may be helpful to construct the best preventive measures.

Effects of dihydroorotate dehydrogenase (DHODH) inhibitors on the growth of Theileria equi and Babesia caballi in vitro.

Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC50) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 µM, respectively, whereas the IC50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 µM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP.

Evaluation of in vitro inhibitory effect of enoxacin on Babesia and Theileria parasites.

Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) structurally related to nalidixic acid used mainly in the treatment of urinary tract infections and gonorrhea. Also it has been shown recently that it may have cancer inhibiting effect. The primary antibabesial effect of Enoxacin is due to inhibition of DNA gyrase subunit A, and DNA topoisomerase. In the present study, enoxacin was tested as a potent inhibitor against the in vitro growth of bovine and equine Piroplasms. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by micro molar concentrations of enoxacin (IC50 values = 33.5, 15.2, 7.5 and 23.2 µM for Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, respectively). Enoxacin IC50 values for Babesia and Theileria parasites were satisfactory as the drug is potent antibacterial drug with minimum side effects. Therefore, enoxacin might be used for treatment of Babesiosis and Theileriosis especially in case of mixed infections with bacterial diseases or incase of animal sensitivity against diminazin toxicity.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia 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 in vitro 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 Babesia parasites and 5% for equine Babesia and Theileria 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 in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.