Evaluation of a Fluorescence-Based Method for Antibabesial Drug Screening

Abstract

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 (r 2 ) value. Subsequently, 50% inhibitory concentration (IC 50 ) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC 50 s 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 IC 50 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. Babesiosis, which is caused by Babesia parasites, has a deleterious effect on the health status of cattle (1). Early diagnosis and treatment with effective antiprotozoan agents are essential for the fast recovery of affected animals (2, 3). The development of drug resistance to and toxic side effects of the drugs currently available for treating babesiosis indicate the importance of introducing new alternative treatment options in veterinary practice (4). Potential novel antibabesial agents are usually evaluated in vitro before clinical trials are conducted. At present, the growth-inhibiting effects of the drug candidates are determined based on parasitemia dynamics, which usually involves microscopic examination of Giemsa-stained thin blood smears of treated and untreated parasites (5, 6). However, microscopy that requires good-quality smears is a time-consuming technique and is unsuitable for mass screening of potential drug candidates. In addition, significant differences in parasitemia estimated by different personnel may be found.Incorporation 3 H]isoleucine, has been a common method in in vitro antimalarial drug screening (7,8). This method is slowly being replaced by a fluorescence-based method because the former is costly, requires numerous procedures, and is c...

Biological Activities of Gedunin—A Limonoid from the Meliaceae Family

“…Morphological and degenerative changes were also observed in parasites treated with gedunin. An alternative method was later developed and optimized to microscopy screening of Babesia, a fluorescence-based method [104,105]. The newly developed method for drug evaluation against B. bovis was successfully achieved; the IC 50 value obtained, 19 µM [104], was comparable to the previously published microscopy-based value, 21.7 µM [103], with no statistically significant (p < 0.05) difference between them.…”

“…An alternative method was later developed and optimized to microscopy screening of Babesia, a fluorescence-based method [104,105]. The newly developed method for drug evaluation against B. bovis was successfully achieved; the IC 50 value obtained, 19 µM [104], was comparable to the previously published microscopy-based value, 21.7 µM [103], with no statistically significant (p < 0.05) difference between them. Initially, this method was not suitable for a large scale screening, since medium had to be replaced every day, but that was later overcome [105].…”

Human Babesiosis : A General Review with Special Reference to Egypt

“…They concluded that prevention and control programs against sources and reservoirs of zoonoses should be planned by public health and veterinary officers based on reliable information from systematic surveillance. Guswanto et al (2014) evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen anti-babesial agents in in vitro cultures of Egyptian B. bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r(2)) value.…”

Comparative assessment of the effect of Diminazene aceturate and Imidocarb dipropionate on haematology and serum biochemical parameters of apparently healthy Nigerian dogs.

“…The main aim of the synthesis of this compound was to produce a chemical that would be used in combating diseases caused by blood parasite infections such as Trypanosoma sp and Babesia sp [3]. DIAC has been utilized in as the main trypanocidal and babesicidal drug in different regions of the world because of its excellent therapeutic index [4,5,6,7]. Even though DIAC has been widely studied over the last 60 years, there have been extensive studies in recent times on it owing to its therapeutic potentials and pleiotropic effects [3,8].…”