Investigation Of Ticks And Five Tick-borne Pathogens Infections In Selected Areas Of China And The Screening Of Nine Acaricidal And Repellent Traditional Chinese Medicine

Ticks are specialist parasitic arthropods that can parasitise all vertebrates,including domestic animals,wildlife and humans.The main threat to humans and animals is that ticks can act as vectors for a wide range of pathogens,mainly viruses,bacteria,protozoa and worms.In the past few decades,due to changes in the climatic environment,tick populations are increasing worldwide and their geographical range is expanding,raising global concerns.Understanding the geographical distribution of tick species and the prevalent tick-borne pathogens is essential for effective surveillance and control of tick-borne diseases.In addition,chemical acaricides are currently the main means of tick control,but their indiscriminate and repeated use has led to the development of acaricide resistance,the accumulation of chemical residues in food,and also has a negative impact on the environment.Therefore,understanding the distribution of tick species and the prevalence of tick-borne pathogens,and screening green and safe tick repellents are important for the development of effective and eco-friendly alternatives for tick control,and can also provide some reference for the control of ticks and tick-borne pathogens in China.1.Identification of tick species in selected areas of ChinaMorphological methods and molecular biology were used to identify species from 2749 ticks,521 groups of tick pool samples collected from seven locations in Jiaozuo,Jiyuan,Xinyang,Zhengzhou,Henan Province,Haikou,Hainan Province,Linyi,Shandong Province and Harbin,Heilongjiang Province from April 2021 to June 2022.The results showed that a total of five tick species were identified at the seven sites,namely Haemaphysalis longicornis,Rhipicephalus microplus,Haemaphysalis hystricis,Dermacentor nuttalli and Haemaphysalis flava,with H.longicornis being the dominant tick species,detected in all five locations except Zhengzhou and Haikou.The next most widely distributed species was the R.microplus,which was detected in Jiyuan and Haikou,the H.hystricis and the D.nuttalli in Xinyang and Harbin respectively,and the H.flava in Zhengzhou,where the H.hystricis was the first to be investigated in Henan.H.longicornis was detected in goats,cattle and dogs as well as in free ticks,D.nuttalli was detected in goats in Harbin,most of the ticks infecting cattle were R.microplus,H.flava was detected in dogs in addition to H.longicornis,and the tick species infecting wild boars was H.hystricis.This study has identified the main tick species prevalent in some areas of China and the main tick species infecting some hosts,providing a reference for accurate tick prevention and control.2.Investigation on the prevalence of five tick-borne pathogens in selected areas of ChinaThe PCR method was used to test 521 tick pool samples from seven locations in Jiaozuo,Jiyuan,Zhengzhou and Xinyang,Henan Province,Haikou,Hainan Province,Linyi,Shandong Province and Harbin,Heilongjiang Province for Piroplasmida,Anaplasma phagocytophilum,Anaplasma ovis,Anaplasma bovis and Bartonella spp.were tested.The results showed that the total positive rate for tick-borne pathogens was 19.19%(100/521),with the highest rate of 10.94%(57/521)for A.bovis,followed by 6.91%(36/521)for A.ovis,3.84%(20/521)for A.phagocytophilum and 1.34%(7/521)for Piroplasmida.No Bartonella spp.were detected in this survey.There were significant differences in tick-borne pathogens in different regions(P<0.05).The tick-borne pathogens in Haikou,Jiaozuo,Xinyang,Linyi,Zhengzhou,Harbin and Jiyuan were 45.83%(44/96),26.47%(18/68),20.48%(17/83),16.18%(11/68),14.29%(1/7),13.11%(8/61),and 0.72%(1/138),respectively.There were significant differences in the pathogens carried by ticks at different physiological stages(P<0.05),with 26.30%(71/270)of adult ticks carrying significantly more pathogens than 11.84%(29/245)of nymphs,0%(0/3)of larvae and 0%(0/3)of eggs.There were significant differences in the pathogen carrying status of ticks from different hosts(P<0.05),with 37.14%(13/35)in wild boar > 34.75%(49/141)in cattle > 21.05%(8/38)in dogs > 9.76%(29/297)in goats.There were significant differences in the pathogens carried by different species of ticks(P<0.05),with 37.14%(13/35)of H.hystricis > 34.92%(44/126)of R.microplus > 14.29%(1/4)of H.flava > 12.70%(39/307)of H.longicornis > 6.52%(3/46)of D.nuttalli.Tick-borne pathogens were mostly mixed infection,and the mixed infection rate of two or more infections was 3.65%(19/521).The present study has identified the carriage of ticks and tick-borne pathogens in some areas of China and provides a reference for the prevention and control of tick-borne diseases.3.Evaluation of insecticidal and repellent effects of ethanol extracts of nine Chinese herbal medicines on R.microplus larvaeIn this study,90% ethanol was used to extract star anise(Illicium verum),Mandarin orange peel(citri reticulatae pericarpium(CRP)),chaulmoogra(Hydnocarpus anthelmintica),stemona(Stemona sessilifolia),motherwort(Leonurus cardiac),clove(Syzygium aromaticum),castor bean(Ricinus communis),shrubby sophora(Sophora flavescens)and box bean(Entada phaseoloides).The insecticidal activity of nine Chinese herbal medicines against larvae and eggs of R.microplus and the repellent activity against larvae of R.microplus were evaluated by fumigation method,filter paper membrane method and insect immersion method.The insecticidal results showed that the ethanolic extracts of star anise,chaulmoogra,motherwort,CRP and stemona had better thixotropic activity against ticks with 100.00%,98.00%,94.00%,88.00% and 86.00%,respectively.The ethanolic extracts of star anise and clove showed better fumigant tick-killing activity with98.00% and 86.00%,respectively.The inhibition of egg hatching by star anise against eggs of R.microplus reached 100.00%.The repellent results showed that the repellent rate of the nine herbs was the highest at 0 h,with all above 80%.As time increased,their repellency rates all decreased to varying degrees,with only castor bean(81.25%),star anise(79.63%),chaulmoogra(66.27%)and motherwort(66.27%)maintaining repellency rates above 50%after 48 h.The repellency of the ethanolic extracts of the nine herbs increased with increasing concentration,with the highest repellency at 1 g/m L and a linear relationship between repellency and concentration.The ethanolic extract of Clove had the highest repellent activity(RC50= 0.562 g/m L),while the RC50 of the remaining eight herbal alcoholic extracts were all >0.7 g/m L.The results of this first study on the tick-killing activity and repellent activity of these herbs against larvae and eggs of R.microplus suggest that the above drugs could be further investigated as potential alternatives to chemical control drugs.