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Background This study aimed to survey the prevalence, antimicrobial resistance, and virulence-associated genes of Salmonella enterica recovered from broiler chickens and retail shops at El-Sharkia Province in Egypt. Salmonella virulence factors were determined using the polymerase chain reaction assays targeting the invA, csgD, hilC, bcfC, stn, avrA, mgtC, ompF, sopE1 and pefA genes. Results One hundred tweenty out of 420- samples from broiler chickens’ cloacal swabs, farm environmental samples, and freshly dressed whole chicken carcasses were positive Salmonella species. The isolates were serotyped as S. Enteritidis as the most dominant serotypes . Interestingly, none of the isolates were resistant to imipenem. The multidrug resistance was determined in 76.7% of the isolates with multidrug antibiotic resistance index of 0.2–0.6. Eight virulence genes ( invA, csgD, hilC, stn, bcfC, mgtC, avrA, and ompf ) were characterized among 120  S. enterica isolates with variable frequencies, while sopE1 and pefA genes that were completely absent in all isolates. Based on the combination of presence and absence of virulence genes, the most common genetic profile (P7, 30%) was invA and csgD genes. Conclusion S . Enteritidis and S . Typhimurium were the most common identified serotypes in the examined sources. Circulation of such strains in broiler farms required introducing special biosecurity and biocontrol measures for control of Salmonella . Such measures might limit the adverse effects of antibiotics and ensure the safety of the environment and animal-derived food.

Background Buffaloes are important contributors to the livestock economy in many countries, particularly in Asia, and tick-borne pathogens (TBPs) commonly infect buffaloes, giving rise to serious pathologies other than their zoonotic potential. Methods The present investigation focuses on the prevalence of TBPs infecting buffaloes worldwide. All published global data on TBPs in buffaloes were collected from different databases (e.g., PubMed, Scopus, ScienceDirect, and Google Scholar) and subjected to various meta-analyses using OpenMeta[Analyst] software, and all analyses were conducted based on a 95% confidence interval. Results Over 100 articles discussing the prevalence and species diversity of TBPs in buffaloes were retrieved. Most of these reports focused on water buffaloes ( Bubalus bubalis ), whereas a few reports on TBPs in African buffaloes ( Syncerus caffer ) had been published. The pooled global prevalence of the apicomplexan parasites Babesia and Theileria , as well as the bacterial pathogens Anaplasma , Coxiella burnetii , Borrelia , Bartonella , and Ehrlichia in addition to Crimean-Congo hemorrhagic fever virus, were all evaluated based on the detection methods and 95% confidence intervals. Interestingly, no Rickettsia spp. were detected in buffaloes with scarce data. TBPs of buffaloes displayed a fairly high species diversity, which underlines the high infection risk to other animals, especially cattle. Babesia bovis , B. bigemina , B. orientalis , B. occultans and B. naoakii , Theileria annulata , T. orientalis  complex ( orientalis/sergenti/buffeli ), T. parva , T. mutans , T. sinensis , T. velifera , T. lestoquardi -like, T. taurotragi , T. sp. (buffalo) and T. ovis , and Anaplasma marginale , A. centrale , A. platys , A. platys -like and “ Candidatus Anaplasma boleense” were all were identified from naturally infected buffaloes. Conclusions Several important aspects were highlighted for the status of TBPs, which have serious economic implications for the buffalo as well as cattle industries, particularly in Asian and African countries, which should aid in the development and implementation of prevention and control methods for veterinary care practitioners, and animal owners. Graphical Abstract

Background Developing new antibabesial drugs with a low toxic effect to the animal and with no resistance from Babesia parasites is in urgent demand. In this concern, the antimalarial, anticancer and antioxidant effect of thymoquinone (TQ), a phytochemical compound found in the plant Nigella sativa , has been reported. Therefore, in the present study, the antibabesial effect of this compound was evaluated on the growth of piroplasm parasites. Results Significant inhibition ( P < 0.05) of the in vitro growth of piroplasm parasites were observed after treatment by TQ with IC 50 values of 35.41 ± 3.60, 7.35 ± 0.17, 0.28 ± 0.016, 74.05 ± 4.55 and 67.33 ± 0.94 μM for Babesia bovis , Babesia bigemina , Babesia divergens , Theileria equi and Babesia caballi , respectively. The in vitro inhibitory effect of TQ was significantly enhanced ( P < 0.05) when used in combination with either diminazene aceturate on bovine Babesia and equine Babesia and Theileria cultures. In B. microti -infected mice, oral and intraperitoneal administrations of TQ showed significant ( P < 0.05) inhibition of parasite growth at a dose of 70 mg/kg and 50 mg/kg, respectively, compared to the control group. Conclusions The obtained results indicate that thymoquinone might be a promising medicinal compound for use in the treatment of animal piroplasmosis.

In this study, we designed novel truncated Babesia caballi ( B . caballi ) recombinant proteins from the previously used B . caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B . caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and a half of each antigen in the cocktail formulas. The serum samples were collected from different endemic areas in addition to the sera collected from horses experimentally infected with B . caballi were used in the present study. Cocktail antigen in full dose of (rBC134f + rBC48t) exhibited the highest optical density (OD) values with B . caballi –infected sera and showed the lowest OD values with normal equine sera or B . caballi , and Theileria equi mixed infected sera in comparison with the single antigen. Interestingly, the same cocktail antigen exhibited the highest concordance rate (76.74%) and kappa value (0.79) in the screening of 200 field serum samples collected from five B . caballi endemic countries, including South Africa (n = 40), Ghana (n = 40), Mongolia (n = 40), Thailand (n = 40), and China (n = 40) using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. Moreover, the identified promising cocktail full dose antigen (rBC134f + rBC48t) showed that it can detect the infection as early as the 4 th day post-infection in sera collected from experimentally infected horses. The obtained results revealed the reliability of the rBC134f + rBC48t cocktail antigen when used in full dose for the detection of specific antibodies to B . caballi in horses which will be useful for epidemiological surveys and control of equine babesiosis .

Babesiosis is an infectious disease with an empty drug pipeline. A search inside chemical libraries for novel potent antibabesial candidates may help fill such an empty drug pipeline. A total of 400 compounds (200 drug-like and 200 probe-like) from the Malaria Box were evaluated in the current study against the in vitro growth of Babesia divergens (B. divergens), a parasite of veterinary and zoonotic importance. Novel and more effective anti-B. divergens drugs than the traditionally used ones were identified. Seven compounds (four drug-like and three probe-like) revealed a highly inhibitory effect against the in vitro growth of B. divergens, with IC50s ≤ 10 nanomolar. Among these hits, MMV006913 exhibited an IC50 value of 1 nM IC50 and the highest selectivity index of 32,000. The atom pair fingerprint (APfp) analysis revealed that MMV006913 and MMV019124 showed maximum structural similarity (MSS) with atovaquone and diminazene aceturate (DA), and with DA and imidocarb dipropionate (ID), respectively. MMV665807 and MMV665850 showed MMS with each other and with ID. Of note, a high concentration (0.75 IC50) of MMV006913 caused additive inhibition of B. divergens growth when combined with DA at 0.75 or 0.50 IC50. The Medicines for Malaria Venture box is a treasure trove of anti-B. divergens candidates according to the obtained results.

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.

Ticks and tick-borne pathogens (TTBPs) are listed among the most serious concerns harming Egyptian livestock’s productivity. Several reports on tick-borne pathogens (TBPs) from various geographical regions in the country were published. However, data on the molecular characterization of TBPs are the most beneficial for understanding the epidemiology of this important group of pathogens. In this study, we present the first meta-analysis on the molecular epidemiology and species diversity of TBPs infecting animals in Egypt. All published studies on TBPs were systematically collected from various databases (PubMed, Scopus, ScienceDirect, the Egyptian Knowledge Bank, and Google Scholar). Data from eligible papers were extracted and subjected to various analyses. Seventy-eight studies were found to be eligible for inclusion. Furthermore, ticks infesting animals that were molecularly screened for their associated pathogens were also included in this study to display high species diversity and underline the high infection risk to animals. Theileria annulata was used as parasite model of TBPs to study the genetic diversity and transmission dynamics across different governorates of Egypt. This study extends cross-comparisons between all published molecular data on TBPs in Egypt and provides resources from Egyptian data in order to better understand parasite epidemiology, species diversity, and disease outcome as well as the development and implementation of prevention and control methods for public health, veterinary care practitioners, and animal owners all over the country.

In this study, we evaluated the validity of a fluorescence-based assay using SYBR Green I (SG I) stain for screening antibabesial compounds against B. microti in mice. Two different hematocrits (HCTs; 2.5% and 5%) were used. Correlating relative fluorescence units (RFUs) with parasitemia showed significant linear relationships with R 2 values of 0.97 and 0.99 at HCTs of 2.5% and 5%, respectively. Meanwhile, the Z′ factors in a high-throughput screening (HTS) assay were within the permissible limit (≥0.5) at 2.5% HCT and lower than this value at 5% HCT. Taken together, the highest signal-to-noise (S/N) ratios were obtained at 2.5% HCT; therefore, we concluded that 2.5% was the best HCT for applying fluorescence assay in antibabesial drug screening in mice. Additionally, positive control mice and those treated with diminazene aceturate, pyronaridine tetraphosphate, and an allicin/diminazene aceturate combination showed peak parasitemia and fluorescence values on the same day post-inoculation. Moreover, using different concentrations of SG I revealed that the optimal concentration was 2x. In summary, considering that all experiments were applied under optimal laboratory conditions, fluorescence assay at 2.5% HCT using 2x SG I for B. microti parasite offers a novel approach for drug screening in mice.

Background An innovative approach has been introduced for identifying and developing novel potent and safe anti- Babesia and anti- Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds ( n  = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations. Methods A fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR). Results Screening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant ( P  < 0.05) downregulation of cysteine protease mRNA levels was observed in MMV665875-treated Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID). Conclusions Inhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives. Graphical Abstract

Piroplasmosis, caused by Babesia spp. and Theileria spp., poses significant constraints for livestock production and upgradation in Bangladesh. Besides examining blood smears, few molecular reports are available from some selected areas in the country. Therefore, the actual scenario of piroplasmosis in Bangladesh is deficient. This study aimed to screen the piroplasms in different livestock species by molecular tools. A total of 276 blood samples were collected from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in five geographies of Bangladesh. After that, screening was conducted through a polymerase chain reaction, and species were confirmed by sequencing. The prevalence of Babesia bigemina, B. bovis, B. naoakii, B. ovis, Theileria annulata and T. orientalis was 49.28%, 0.72%, 1.09%, 32.26%, 6.52% and 46.01%, respectively. The highest prevalence (79/109; 72.48%) of co-infections was observed with B. bigemina and T. orientalis. The phylogenetic analyses revealed that the sequences of B. bigemina (BbigRAP-1a), B. bovis (BboSBP-4), B. naoakii (AMA-1), B. ovis (ssu rRNA) and T. annulata (Tams-1) were included in one clade in the respective phylograms. In contrast, T. orientalis (MPSP) sequences were separated into two clades, corresponding to Types 5 and 7. To our knowledge, this is the first molecular report on piroplasms in gayals and goats in Bangladesh.