The aim of this study was to evaluate the comparative changes of selected acute-phase proteins (A... more The aim of this study was to evaluate the comparative changes of selected acute-phase proteins (APPs), alkaline phos-phatase (ALP), and lactate dehydrogenase (LDH) activities in buffalos and cows with subclinical mastitis. For this purpose, a total of 139 buffalos (Bubalus bubalis) and 146 cows (Holstein or Friesian) from Dakahlia governorate in Egypt were studied. Seventy-six cows and 49 buffalos exhibited subclinical mastitis with positive California mastitis test (CMT) and no clinical signs of mastitis. Results revealed that the most dominant isolated bacteria were Staphylococcus spp. in both buffalo and cow fellowed by Streptococcus spp. and E.coli. Statistical significant differences were observed in SCC, HP level, and LDH activity between buffalos with different bacterial isolates. Milk HP level showed significant differences (P < 0.05) between clinically health buffalos and cows and those infected with different isolated pathogen. In conclusion, similar to cows, Hp is a sensitive marker of subclinical mastitis in buffalo, and they may be advantageous to clarify the host response to the inflammation of mam-mary gland in clinical research. However, additional studies on a wider range of APPs and specific pathogens in buffalos are required to explore the response pattern to mastitis in such species.
Host cell invasion is the only step where Babesia parasites are extracellular, and their survival... more Host cell invasion is the only step where Babesia parasites are extracellular, and their survival is menaced during this step. Therefore, interfering with this critical stage is a target for an anti-Babesia intervention strategy. In this regard, recombinant protein encoding Babesia divergens Erythrocyte Binding Protein (BdEBP) was produced in Es-cherichia coli in the current study, and its antiserum was prepared in mice for further molecular characterization. Western blotting and indirect fluorescent antibody test (IFAT) revealed the specific reaction of the anti-rBdEBP serum with a corresponding authentic protein of B. divergens. Next, bovine RBCs were incubated with a B. diver-gens lysate, and anti-rBdEBP serum was produced in mice to detect the ability of BdEBP to bind with host cells. Bands corresponding to 29.6-kDa proteins in the protein-bound erythrocyte lysate were detected by specific immune rBdEBP using Western blotting. These results suggest that BdEBP is functional in the merozoite stage and may be involved in attachment to bovine RBCs. A significant inhibition of the in vitro growth of B. divergens culture treated with anti-rBdEBP serum was observed. Moreover, the efficacy of pre-incubated free merozoites to invade bovine erythrocytes was inhibited by 60% after incubation with 2 mg/ml of anti-rBdEBP serum for 6 h. The obtained data suggest the possible use of rBdEBP as a vaccine candidate against bovine babesiosis.
N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant acti... more N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate.
The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the ... more The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the effica-cies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC 50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence-and microscopy-based methods , did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate-and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (−)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle.
Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) struc... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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.
A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for
the... more 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.
In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become... more In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined by the fluorescence-based method (408 nM and 8.13 M, respectively) and microscopy (400.3 nM and 9.4 M, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 M) was similar to the recently described microscopy-based value (21.7 M) for B. bovis. Additionally, the Z= factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay.
Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivi... more Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood– DNA samples sourced from cattle (n = 439), buffaloes (n = 50), and sheep (n = 105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasiteswas only found in twoindividualDNA samples (both 2%),while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3–100% and 95.3–100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8–100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major PiroplasmSurface Protein (MPSP) gene sequenceswere classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2,which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.
Cytokines play an important role in innate immune
response to diseases in bovine calves. The obje... more Cytokines play an important role in innate immune response to diseases in bovine calves. The objective of the present study was to assess the comparative changes of selected acute-phase proteins (APPs) and immunoglobulins in buffalo and bovine calves with respiratory disease. For this purpose, a total of 65 calves (28 buffalo and 37 bovine) were studied. Of all, 45 calves (21 buffalo and 24 bovine) were diagnosed to have respiratory disease. In addition, 20 clinically healthy calves (7 buffalo and 13 bovine) were randomly selected as a control group. Clinical and hematological examinations as well as measurement of haptoglobin (Hp), fibrinogen (Fb), IgG, IgA, total protein (TP), albumin, and globulin were carried out. Clinically, in acute respiratory disease, buffalo calves had lower clinical index scores (p<0.05) than bovine calves. In both buffalo and bovine calves, Fb and Hp levels were increased significantly (p<0.05) in acute respiratory disease compared with chronic cases. However, IgA, IgG, and albumin levels showed a significant decrease (p<0.05). Moreover, buffalo calves had a significant increase (p<0.05) of Hp and IgG compared with bovine calves. Total protein and globulin levels showed a significant decrease (p<0.05) in calves with chronic respiratory diseases. In conclusion, Hp and IgG response in buffalo calves with respiratory disease seems to differ from bovine calves. Further studies need to be done on experimental respiratory infection in buffalo calves to elucidate the specific acute-phase response.
The aim of this study was to evaluate the comparative changes of selected acute-phase proteins (A... more The aim of this study was to evaluate the comparative changes of selected acute-phase proteins (APPs), alkaline phos-phatase (ALP), and lactate dehydrogenase (LDH) activities in buffalos and cows with subclinical mastitis. For this purpose, a total of 139 buffalos (Bubalus bubalis) and 146 cows (Holstein or Friesian) from Dakahlia governorate in Egypt were studied. Seventy-six cows and 49 buffalos exhibited subclinical mastitis with positive California mastitis test (CMT) and no clinical signs of mastitis. Results revealed that the most dominant isolated bacteria were Staphylococcus spp. in both buffalo and cow fellowed by Streptococcus spp. and E.coli. Statistical significant differences were observed in SCC, HP level, and LDH activity between buffalos with different bacterial isolates. Milk HP level showed significant differences (P < 0.05) between clinically health buffalos and cows and those infected with different isolated pathogen. In conclusion, similar to cows, Hp is a sensitive marker of subclinical mastitis in buffalo, and they may be advantageous to clarify the host response to the inflammation of mam-mary gland in clinical research. However, additional studies on a wider range of APPs and specific pathogens in buffalos are required to explore the response pattern to mastitis in such species.
Host cell invasion is the only step where Babesia parasites are extracellular, and their survival... more Host cell invasion is the only step where Babesia parasites are extracellular, and their survival is menaced during this step. Therefore, interfering with this critical stage is a target for an anti-Babesia intervention strategy. In this regard, recombinant protein encoding Babesia divergens Erythrocyte Binding Protein (BdEBP) was produced in Es-cherichia coli in the current study, and its antiserum was prepared in mice for further molecular characterization. Western blotting and indirect fluorescent antibody test (IFAT) revealed the specific reaction of the anti-rBdEBP serum with a corresponding authentic protein of B. divergens. Next, bovine RBCs were incubated with a B. diver-gens lysate, and anti-rBdEBP serum was produced in mice to detect the ability of BdEBP to bind with host cells. Bands corresponding to 29.6-kDa proteins in the protein-bound erythrocyte lysate were detected by specific immune rBdEBP using Western blotting. These results suggest that BdEBP is functional in the merozoite stage and may be involved in attachment to bovine RBCs. A significant inhibition of the in vitro growth of B. divergens culture treated with anti-rBdEBP serum was observed. Moreover, the efficacy of pre-incubated free merozoites to invade bovine erythrocytes was inhibited by 60% after incubation with 2 mg/ml of anti-rBdEBP serum for 6 h. The obtained data suggest the possible use of rBdEBP as a vaccine candidate against bovine babesiosis.
N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant acti... more N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate.
The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the ... more The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the effica-cies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC 50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence-and microscopy-based methods , did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate-and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (−)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle.
Enoxacin is a broad-spectrum 6-fluoronaphthyridinone antibacterial agent (fluoroquinolones) struc... more 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 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.
A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for
the... more 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.
In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become... more In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined by the fluorescence-based method (408 nM and 8.13 M, respectively) and microscopy (400.3 nM and 9.4 M, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 M) was similar to the recently described microscopy-based value (21.7 M) for B. bovis. Additionally, the Z= factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay.
Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivi... more Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood– DNA samples sourced from cattle (n = 439), buffaloes (n = 50), and sheep (n = 105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasiteswas only found in twoindividualDNA samples (both 2%),while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3–100% and 95.3–100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8–100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major PiroplasmSurface Protein (MPSP) gene sequenceswere classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2,which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.
Cytokines play an important role in innate immune
response to diseases in bovine calves. The obje... more Cytokines play an important role in innate immune response to diseases in bovine calves. The objective of the present study was to assess the comparative changes of selected acute-phase proteins (APPs) and immunoglobulins in buffalo and bovine calves with respiratory disease. For this purpose, a total of 65 calves (28 buffalo and 37 bovine) were studied. Of all, 45 calves (21 buffalo and 24 bovine) were diagnosed to have respiratory disease. In addition, 20 clinically healthy calves (7 buffalo and 13 bovine) were randomly selected as a control group. Clinical and hematological examinations as well as measurement of haptoglobin (Hp), fibrinogen (Fb), IgG, IgA, total protein (TP), albumin, and globulin were carried out. Clinically, in acute respiratory disease, buffalo calves had lower clinical index scores (p<0.05) than bovine calves. In both buffalo and bovine calves, Fb and Hp levels were increased significantly (p<0.05) in acute respiratory disease compared with chronic cases. However, IgA, IgG, and albumin levels showed a significant decrease (p<0.05). Moreover, buffalo calves had a significant increase (p<0.05) of Hp and IgG compared with bovine calves. Total protein and globulin levels showed a significant decrease (p<0.05) in calves with chronic respiratory diseases. In conclusion, Hp and IgG response in buffalo calves with respiratory disease seems to differ from bovine calves. Further studies need to be done on experimental respiratory infection in buffalo calves to elucidate the specific acute-phase response.
Uploads
Papers by mohamed rizk
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.
of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although
microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study
evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia
bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944
goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents,
diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined
by the fluorescence-based method (408 nM and 8.13 M, respectively) and microscopy (400.3 nM and 9.4 M, respectively) were
in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 M) was similar to the recently
described microscopy-based value (21.7 M) for B. bovis. Additionally, the Z= factor (0.80 to 0.90), signal-to-noise (S/N)
ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the
minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable
to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method
might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS)
assay.
greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia
bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood–
DNA samples sourced from cattle (n = 439), buffaloes (n = 50), and sheep (n = 105) reared in Menoufia,
Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and
T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were
the only parasites detected in buffaloes and each of these parasiteswas only found in twoindividualDNA samples
(both 2%),while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively.
Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical
Membrane Antigen-1 genes were highly conserved among the samples, with 99.3–100% and 95.3–100% sequence
identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences
were relatively diverse (87.8–100% identity values), dispersing themselves across several clades in the
phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major PiroplasmSurface
Protein (MPSP) gene sequenceswere classified as types 1 and 2. This is the first report of T. orientalis in Egypt,
and of type 2 MPSP in buffaloes. Detection of MPSP type 2,which is considered a relatively virulent genotype, suggests
that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present
study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may
shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.
response to diseases in bovine calves. The objective of the
present study was to assess the comparative changes of selected
acute-phase proteins (APPs) and immunoglobulins in
buffalo and bovine calves with respiratory disease. For this
purpose, a total of 65 calves (28 buffalo and 37 bovine) were
studied. Of all, 45 calves (21 buffalo and 24 bovine) were
diagnosed to have respiratory disease. In addition, 20 clinically
healthy calves (7 buffalo and 13 bovine) were randomly
selected as a control group. Clinical and hematological examinations
as well as measurement of haptoglobin (Hp), fibrinogen
(Fb), IgG, IgA, total protein (TP), albumin, and globulin
were carried out. Clinically, in acute respiratory disease, buffalo
calves had lower clinical index scores (p<0.05) than
bovine calves. In both buffalo and bovine calves, Fb and Hp
levels were increased significantly (p<0.05) in acute respiratory
disease compared with chronic cases. However, IgA,
IgG, and albumin levels showed a significant decrease
(p<0.05). Moreover, buffalo calves had a significant increase
(p<0.05) of Hp and IgG compared with bovine calves. Total
protein and globulin levels showed a significant decrease
(p<0.05) in calves with chronic respiratory diseases. In conclusion,
Hp and IgG response in buffalo calves with respiratory
disease seems to differ from bovine calves. Further studies
need to be done on experimental respiratory infection in
buffalo calves to elucidate the specific acute-phase response.
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.
of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although
microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study
evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia
bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944
goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents,
diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined
by the fluorescence-based method (408 nM and 8.13 M, respectively) and microscopy (400.3 nM and 9.4 M, respectively) were
in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 M) was similar to the recently
described microscopy-based value (21.7 M) for B. bovis. Additionally, the Z= factor (0.80 to 0.90), signal-to-noise (S/N)
ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the
minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable
to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method
might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS)
assay.
greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia
bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood–
DNA samples sourced from cattle (n = 439), buffaloes (n = 50), and sheep (n = 105) reared in Menoufia,
Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and
T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were
the only parasites detected in buffaloes and each of these parasiteswas only found in twoindividualDNA samples
(both 2%),while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively.
Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical
Membrane Antigen-1 genes were highly conserved among the samples, with 99.3–100% and 95.3–100% sequence
identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences
were relatively diverse (87.8–100% identity values), dispersing themselves across several clades in the
phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major PiroplasmSurface
Protein (MPSP) gene sequenceswere classified as types 1 and 2. This is the first report of T. orientalis in Egypt,
and of type 2 MPSP in buffaloes. Detection of MPSP type 2,which is considered a relatively virulent genotype, suggests
that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present
study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may
shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt.
response to diseases in bovine calves. The objective of the
present study was to assess the comparative changes of selected
acute-phase proteins (APPs) and immunoglobulins in
buffalo and bovine calves with respiratory disease. For this
purpose, a total of 65 calves (28 buffalo and 37 bovine) were
studied. Of all, 45 calves (21 buffalo and 24 bovine) were
diagnosed to have respiratory disease. In addition, 20 clinically
healthy calves (7 buffalo and 13 bovine) were randomly
selected as a control group. Clinical and hematological examinations
as well as measurement of haptoglobin (Hp), fibrinogen
(Fb), IgG, IgA, total protein (TP), albumin, and globulin
were carried out. Clinically, in acute respiratory disease, buffalo
calves had lower clinical index scores (p<0.05) than
bovine calves. In both buffalo and bovine calves, Fb and Hp
levels were increased significantly (p<0.05) in acute respiratory
disease compared with chronic cases. However, IgA,
IgG, and albumin levels showed a significant decrease
(p<0.05). Moreover, buffalo calves had a significant increase
(p<0.05) of Hp and IgG compared with bovine calves. Total
protein and globulin levels showed a significant decrease
(p<0.05) in calves with chronic respiratory diseases. In conclusion,
Hp and IgG response in buffalo calves with respiratory
disease seems to differ from bovine calves. Further studies
need to be done on experimental respiratory infection in
buffalo calves to elucidate the specific acute-phase response.