Vaccine development based on gene-edited parasite of Neospora caninum and identification of drug candidates from wild medical plants for control of protozoan infection

概要

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apic omplexan parasite. Dogs are its definitive host, it mostly infects livestock a nimals, especially cattle that acts as an intermediate host. It is necessary t o have well-established models of abortion and vertical transmission in exp erimental animals, in order to determine basic control measures for the N. caninum infection. I evaluated the role of N. caninum dense granule antige n 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/ 6 pregnant mouse model. I inoculated mice on day 3.5 of pregnancy with parental Nd or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem ana lyses were performed on day 30 after birth and the surviving pups were k ept until day 30 postpartum. The number of parasites in brain tissues of of fspring from NcGRA7KO-infected dams was significantly lower than that of the Nc1-infected dams under two infection doses (1X106 and 1X105 tachy zoites/mouse). The vertical transmission rates in the NcGRA7KO-infected gr oup were significantly lower than those of the Nd-infected group. To under stand the mechanism by which the lack of NcGRA7 decreased the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection). Although parasite DNA was detected in the placenta s, no significant difference was found between the two parasite lines. Histo pathological analysis revealed a greater inflammatory response in the place ntas from NcGRA7KO-infected dams than in those from the parental strain.

This finding correlated with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7 KO-infected m ice. In conclusion, these results suggest that loss of NcGRA7 triggers an i nflammatory response in the placenta, resulting in decreased vertical trans mission of N. caninum.

Live vaccination is the most protective method against bovine neosporosis, which is the major cause of bovine abortion globally. Here, parenteral stai n Nd and less virulent NcGRA7KO line were evaluated as potential live v accines. Pregnant and non-pregnant BALB/c mice were subcutaneously ino culated with high (1X105) or low (1X104) doses of tachyzoites. Non-pregn ant mice were challenged intraperitoneally with 2X106 Nc1-GFP tachyzoites at 45 days post-vaccination (dpv), and postmortem examination was perfor med at 30 days post-challenge infection (76 dpv). At both doses, NcGRA7 KO-vaccinated animals showed higher bodyweight gain and fewer clinical si gns than unvaccinated animals. Although there was no significant differenc e in the survival rate among experimental groups, the parasite burden in t he brain of vaccinated animals was lower than that of unvaccinated animal s. For pregnancy conditions, mating was started at 3 weeks post-vaccinatio n, and challenge was performed with 1X105 Nc1-GFP at day 10.5 of preg nancy. Postmortem examination was performed at 30 days postpartum. For high-dose vaccination, there was no significant difference in the survival r ates of offspring between vaccinated (27.8%) and unvaccinated dams (39. 1%), whereas for low-dose vaccination, the offspring from vaccinated dams had a significantly higher survival rate (75.0%) than those from unvaccinat ed dams (6.9%). The amount of challenge (Nc1-GFP) parasite DNA in the brain of offspring from vaccinated dams was significantly lower than that of offspring from unvaccinated dams. These results suggest that NcGRA7KO parasites have potential for use as a live attenuated vaccine against N. ca ninum infection.

Since there is no available chemotherapy against different protozoan infecti ons as toxoplasmosis, Neosporosis and malaria, treatment with medicinal pl ants will help to overcome the drug toxicity and parasite resistance compar ed with the commercially used drugs. Wild medicinal plants from the desert have been traditionally used as antimicrobial agents in Egypt. However, litt Ie is known about the antiprotozoal efficacy of these plants. Here, I evaluat ed the in vitro activity of extracts from certain wild Egyptian desert plants against Toxoplasma gondii and N. caninum. Of the 12 plant extracts teste d, the methanolic extracts from Artemisia judaica, Cleome droserifolia, Trich odesma africanum, and Vachellia tortilis demonstrated potent activity agains t the growth of T gondii, with half-maximal inhibitory concentrations 50 (IC5 0) of 2.1 pg/ml, 12.5 pg/ml, 21.8 pg/ml, and 24.5 pg/ml, respectively. Their mean selectivity index (SI) values were 150.8, 29.6, 18.9, and 22.6, respec tively. These extracts, along with an ethanolic extract of P. undulata, were t hen evaluated against N. caninum. C. droserifolia, an ethanolic extract of P. undulata, T africanum, A. judaica, and V. tortilis demonstrated potent eff icacy against the growth of N. caninum in vitro, with mean IC50S of 1.0 pg/ ml, 3.0 pg/ml, 3.1 pg/ml, 8.6 pg/ml, and 17.2 pg/ml, respectively. Their mea n SI values were 370.9, 18.5, 133.2, 36.8, and 32.2, respectively. These fi ndings indicate that A. judaica is most potent against T gondii, while both C. droserifolia and T africanum extracts were more effective to N. caninu m. Our data suggest these extracts could provide an alternative treatment f or T gondii and N. caninum infections.

Finding a novel treatment for malaria is still challenging, and various extra cts from different wild desert plants have been reported to have multiple m edicinal uses for human public health, in my study, I had evaluated the an timalarial efficacy of several Egyptian plant extracts. I assessed the cytotoxi c potential of 13 plant extracts and their abilities to inhibit the in vitro grow th of Plasmodium falciparum (3D7), and to treat infection with non-lethal PI asmodium yoelii 17XNL in an in vivo malaria model in BALB/c mice. In vitr o screening identified four promising candidates, T africanum, A. judaica, C. droserifolia, and V. tortilis, with weak-to-moderate activity against P. falci parum erythrocytic blood stages with mean IC50 of 11.7 pg/ml, 20.0 pg/ml, 32.1 pg/ml, and 40.0 pg/ml, respectively. Their mean SI values were 35.2, 15.8, 11.5, and 13.8, respectively. Morphological alterations, such as cell s hrinkage, and parasite fragmentation were observed following treatment wit h plant extract concentration of 50 pg/ml as well as after treatment with th e positive control drug, chloroquine, in comparison with untreated parasites.

Among these four candidates, T africanum crude extract exhibited the hig hest parasite suppression in a murine malaria model against P. yoelii. As it causes the significantly reduced level of parasitemia observed from 6-15 days post-infection after oral treatment. My study identified novel natural an timalarial agents of plant origin that have the potential for development into therapeutics for treating malaria.