Screening And Application Of The Active Components Of Traditional Chinese Medicine Against Canine Babesia Gibsoni Infection

Babesiosis is a severe blood protozoan disease caused by Babesia,which parasites in the red blood cells of animals.The affected animals are showed by elevated body temperature,rapid weight loss,anemia and hemoglobinuria.The number of red blood cells and the content of hemoglobin are reduced to 45%and 39%,respectively.If the affected dogs are not treated in time,they will die in 4-8 days,with a mortality rate of 50%.Chronic cases can only recover within weeks or months.Therefore,this disease causes great economic losses to the dog-raising industry.Due to lack of safe and effective vaccines,the prevention and treatment of babesiosis mainly depends on drugs.The non-standard clinical use of common drugs used for the treatment of babesiosis causes widespread drug resistance,which brings great difficulties to the treatment of babesiosis.Some drugs are not conducive to long-term use because of toxicity to the body and other factors.Therefore,there is an urgent need to develop anti-Babesia drugs with high efficiency,low toxicity and no residue.In this study,the animal model of artificial infection with Babesia gibsoni was established to observe the effect of Verbena extract against Babesia gibsoni.Then six anti-malaria parasite active components were selected from traditional Chinese medicine for the treatment of malaria,and detected for against Babesia gibsoni activity.Finally,catechin was selected as anti-Babesia gibsoni drug,and its therapeutic effect on babesiosis was observed.1.Changes of clinical symptoms and blood parameters in infected dogs with Babesia gibsoniHealthy dogs were intravenous inoculated with blood samples(5 mL/dog),which were collected from dogs infected with Babesia gibsoni.After inoculation,the clinical symptoms were observed every day,and the blood samples were collected every three days.The infection was detected by blood smear and PCR.Blood routine,blood biochemistry and other clinicopathological indexes before and after infection were detected.The results showed that 9 days after artificial infection,the dogs began to show depression,anorexia and pale mucosa.One dog died on 25 days after infection.Necropsy of dog showed that pleural effusion,ascites and mucosa yellow staining,and extremely enlarged spleen.The specific gene fragments of Babesia gibsoni could be detected by PCR 6 days after infection.RBC,HGB and HCT began to decrease significantly 12 days after infection.The lowest RBC,HGB and HCT were 38.8%,38.9%and 39.9%of pre-infection,respectively.RDW-CV and RDW-SD increased gradually.There was no significant change in white blood cell and other related indexes.In the blood biochemical indexes,TP and ALB decreased gradually,and TBIL and DBIL increased.The results showed that the changes of clinical symptoms and blood indexes were similar between in artificially and naturally infected dogs,and they all showed hemolytic anemia.2.Observation on therapeutic effect of Verbena officinalis extract on Babesia gibsoniVerbena officinalis extract 1 g/mL was prepared by decoction.Verbena officinalis extract(final concentration 1 mg/mL,0.1 mg/mL,0.01 mg/mL)was added to the red blood cell suspension containing Babesia gibsoni and incubated at 37℃ for 24 hours.Relative quantitative real-time PCR was used to evaluate the efficacy of Verbena officinalis extract in killing Babesia gibsoni in vitro by detecting the relative number of 18S rRNA of Babesia gibsoni.When RBC reduced to 2×1012/L after artificial infection with Babesia gibsoni,the Verbena officinalis extract was administered continuously at a dose of 0.45 g/kg for 7 d.The therapeutic effect was determined by observing clinical symptoms and detecting blood routine indexes.The results showed that Verbena officinalis extract of 1 mg/mL had a good effect on killing Babesia gibsoni.Compared with the control group,In Verbena officinalis extract group,the expression of 18S rRNA of Babesia gibsoni decreased by 42%.Diminazene positive drug control group decreased by 57%.When Verbena officinalis extract was used to treat artificially infected dogs after 22 d of treatment,RBC,HGB and HCT increased gradually and recovered to 3.5×1012/L,83 g/L and 25.3%respectively,which were close to the normal level.RBC,HGB and HCT in the diminazene drug positive control group were 4.21×1012/L,94 g/L and 31.2%respectively,and the therapeutic effect was slightly better than Verbena extract treatment.The results indicated that Verbena extract treatment had a good therapeutic effect on Babesia gibsoni,but it was slightly lower than diminazene,which was commonly used in veterinary clinic.3.Screening of active components of traditional Chinese medicine against Babesia gibsoniThe red blood cell suspension containing Babesia gibsoni was treated with verbenoside,catechin,dihydrolycorine,sinomeninol,ursolic acid and brucea javanica H at the concentration of 2 μmol/L,and incubated at 37℃ for 24 h.RNA was extracted and the relative expression of 18S rRNA was detected by quantitative real-time PCR.The red blood cell suspension containing Babesia gibsoni was treated with different concentrations of traditional Chinese medicine.The relative expression of 18S rRNA of Babesia gibsoni was detected after the action of active components of traditional Chinese medicine at different concentrations.The correlation between logarithm of drug concentration and inhibition rate was analysed to obtain the regression formula and calculate IC50.The results showed that the six active ingredients of traditional Chinese medicine had killing effect on Babesia gibsoni.The inhibition rate of catechin at a concentration of 200 nmol/L to was 47%,followed by sinomeninol(43%)and dihydrolycorine(40%).The IC50 of catechin was 273 nmol/L,followed by sinomeninol(327 nmol/L),dihydrolycorine(378 nmol/L),brucea javanica H(451 nmol/L),verbenoside(1091 nmol/L),ursolic acid(1531 nmol/L).The effect of catechin is better than the diminazene(449 nmol/L),which was commonly used in veterinary clinic.4.Safety test and therapeutic effect of catechinThe in vitro test verified that catechin had a good effect on against Babesia gibsoni,and further verified the effect of catechin in the treatment of babesiosis in dogs in vivo.ICR mice were intraperitoneally injected with different concentrations of catechin(2000 mg/kg,1000 mg/kg,500 mg/kg,250 mg/kg,100 mg/kg and 0 mg/kg)to observe the safety of catechin.When RBC of dogs artificially infected with Babesia gibsoni was reduced to about 2×1012/L,catechin(11 mg/kg)was injected intravenously and again after 3 days.The treatment effect was determined by observing the clinical symptoms,detecting the infection rate of red blood cells and the blood routine indexes.The results showed that no mice died after intraperitoneal injection of all doses of catechin,only 2000 mg/kg mice showed shortness of breath after injection and returned to normal after 3 hours.There were no obvious abnormal dissection and histopathological changes in each group.After catechin and diminazene treatment,the blood routine indexes of the affected dogs gradually returned to normal.After 30 d of treatment,the RBC was 4.02×1012/L and 4.06×1012/L in catechin and diminazene treatment.groups,HGB was 108 g/L and 111 g/L,and HCT was 31.8%and 31.9%,respectively.RBC,HGB,and HCT were 2.60×1012/L,82.20 g/L and 22.0%in control group,respectively.Although the clinical symptoms returned to normal,the blood routine indexes did not return to the normal level,and the parasitemia level remained at 0-1/HPF.No dogs died after treatment,and the clinical cure rate was 100%.The therapeutic effects of catechin and diminazene were similar,and there was no significant difference in blood routine and red blood cell infection rate.The results indicated that catechin had a good effect on killing Babesia gibsoni and had a broad application prospect in the treatment of animal babesiosis.