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Evaluation of Doxycycline Sensitivity in Vitro Against Babesia Gibsoni by Real Time-pcr

Dr. Md. Hasanurzzaman Talukder
Professor
Department of Parasitology, Bangladesh Agricultural University, Mymensingh-2202Bangladesh

Abstract/ Executive Summary:

Doxycycline sensitivity against in vitro cultured Babesia gibsoni was evaluated by real-time PCR and parasitemia. The culture of B. gibsoni was successfully continued and mean parasitemia was 4.63% when in vitro drug sensitivity test was started. The drug sensitivity test by the real-time PCR calculated the gene copy number from cultured sample. Even if the complete growth inhibition was at certain concentration, the genomic DNA might remain in existence. The results revealed that the doses of doxycycline 8µM, 16 µM, 32 µM and 64 µM of both real-time PCR and parasitemia inhibited the growth of B. gibsoni in a dose-dependent manner at 96 h and 144 h. We determined the IC50 of doxycycline 17.9 µM at 96 h and 13.8 µM at 144 h after treatment using real-time PCR. Morphological observation revealed that the number of parasites decreased per erythrocyte and line shaped parasites increased in erythrocytes with the increased concentration of doxycycline. Doxycycline effectively inhibits the growth of B. gibsoni in vitro. Therefore, doxycycline can be used to treat B. gibsoni infection. Further studies are important to know the doxycycline resistance in B. gibsoni.

Keywords: Doxycycline, Babesia gibsoni, RT-PCR, Growth inhibition

Location: Mymensingh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

To evaluate doxycycline sensitivity in vitro against Babesia gibsoni by Real Time-PCR

Methodology:

Test compound: Doxycycline hydrochloride was purchased from Sigma-Aldrich (Tokyo, Japan). Different doses of doxycycline (DXC) 2µM, 4µM, 8µM, 16µM, 32µM and 64µM were used to evaluate the growth inhibitory effects. Packed cell volume and thrombocyte count: Measurements of packed cell volume (PCV) and thrombocyte counts were performed using an automated blood cell counter (Celltaq MEK6400, Nihon Kohden Co, Tokyo, Japan). Culture of B. gibsoni: B. gibsoni parasites were isolated from a naturally infected Tosa dog in Aomori Prefecture, Japan (Matsuu et al., 2004a). The 18S rDNA sequences of this parasite exhibit 100% homology with the genotype of B. gibsoni isolated from dogs in Asia, Oklahoma, North Carolina and Okinawa (Matsuu et al., 2004b). The parasite was maintained in the laboratory by passage in beagle dogs. Preparation of erythrocytes from the infected blood sample for culture:The blood sample was washed 3 times with phosphate-buffered saline (PBS) by centrifugation at 4 × g for 10 min at 4 °C. The culture medium was RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 25 mM pyruvic acid, 2 mM l-glutamine, 100 units/mL penicillin G, 100 μg/mL streptomycin (Invitrogen) and 10% fetal bovine serum (FBS; Funakoshi, Tokyo, Japan). Canine erythrocytes sufficient to yield a packed cell volume (PCV) of 10% were obtained from a healthy Beagle. Packed infected RBCs (200 μl) were dispensed into 1800 μl of culture medium in each well of a 12-well plate. This plate was incubated at 37 °C in a humidified atmosphere containing 5% CO₂ and 5% O₂ for 7 days. The supernatant medium (800 μl) was replaced daily by a fresh medium. Sub-culturing was performed 7 days after primary culturing was initiated. Normal fresh erythrocytes were sampled from normal beagles by using sodium citrate as the anticoagulant and immediately washed 3 times with PBS. We transferred 200 μl of the cultured and infected erythrocyte suspension to a new well containing 1620 μl of fresh medium and 180 μl of a normal dog erythrocyte suspension. Subsequent continuous culturing was performed every 5–7 days. The percent parasitemia at subculture was calculated by enumeration of 2000 total infected and uninfected RBC on Giemsa-stained smears under microscope. The ensuing parasitemia was monitored daily as above. Growth inhibitory test of doxycycline: For the in vitro sensitivity test, B. gibsoni cultures that had reached parasitemia approximately 5% (parasitemia before adding drug = Pre ) after the last subculture for 2-3 days were collected in a collection tube. Two hundred mL of these parasitized erythrocyte suspension was distributed per well in 96-well plates in three replicates of each drug concentration. Doxycycline hydrochloride was dissolved in ultra pure water and aliquot was prepared and then added to the culture medium to give the final concentrations of 2µM, 4 µM, 8 µM, 16 µM, 32 µM and 64 µM. This range of drug concentrations were based on the results of preliminary assays conducted at the laboratory. From the aliquots drug was added into each well of the serially diluted doxycycline in culture medium and one well without drug (ND) as control for normal growth of parasite. The plates were incubated in a 5% CO2 incubator at 37⁰C. Every 24 h the medium (80 µL) in each well was aspirated and a fresh solution containing the appropriate concentration of the test drug was added. After 48, 72, 96 and 144 h of incubation from each well 1 µL sample was taken for parsitemia and 1 µL for RT-PCR. Real-Time PCR: DNA was extracted using DNA extraction kit (QIAamp DNA Mini Kit; Qiagen, USA) and Real-Time PCR was performed using a standard protocol recommended by the manufacturer (iQ SYBR Green Supermix, Bio-Rad, Japan). One µL of template DNA was added to 19 µL of reaction mixture containing 0.5 µL of each primer, 8 µL ultra pure water, and 10 µL iQ SYBR Green Supermix. The reaction was performed under the following conditions (Mini Opticon, Bio-Rad) 95⁰C for 30s. 95⁰C for 3min, 55⁰C for 5s. A thermal melt profile was built following PCR to identify amplicons as specified, and analysis was performed with analysis software (CFX-Manager, Bio-Rad). The IC₅₀ of doxycycline was defined as the concentration required for the 50% reduction in the mean number of parasitized erythrocytes from that of control culture after 96 and 144 h of incubation. Each test was performed in triplicates. IC50 values were expressed as the mean concentration ±SD. Parasitemia: A thin smear was prepared, fixed with methanol and stained with Giemsa solution to count the number of parasitized erythrocytes. Parasitemia was monitored by counting the number of parasitized cells among 2000 erythrocytes and expressing this as a percentage. The sensitivity of doxycycline was evaluated by measuring the rate of growth inhibition. This was calculated by counting the number of parasitized erythrocytes per 2000 erythrocytes in each of the wells containing the drug and that in the control well without drug (ND).

Source of Information: J. Bangladesh Agril. Univ. 11(1): 97–102, 2013, ISSN 1810-3030

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The study determined the IC50 of doxycycline 17.9 µM at 96 h and 13.8 µM at 144 h after treatment using real-time PCR. Morphological observation revealed that the number of parasites decreased per erythrocyte and line shaped parasites increased in erythrocytes with the increased concentration of doxycycline. Doxycycline effectively inhibits the growth of B. gibsoni in vitro. Therefore, doxycycline can be used to treat B. gibsoni infection. Further studies are important to know the doxycycline resistance in B. gibsoni. It is concluded that treatment of canine babesiosis by oral administration of doxycycline can be of practical importance. Further studies are important to evaluate the doxycycline resistance gene in B. gibsoni.

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