| Ascariasis of swine is an important intestinal parasitic disease and affects the pig industry seriously in China and the world.It is distributed worldwide.In this study,we first did epidemiological studies of Ascaris suum of the "Hainan black pig",a local breeder in Hainan.The purpose was to get the temporal and spatial distribution pattern of Ascaris suum eggs in soil and water;analyze the relationship between the prevalence of swine ascariasis and farming type,tropical climate,regional environment and social and cultural factors;provide insight into establishing a comprehensive prevention and control program for swine ascariasis,suitable for local pigs in Hainan's tropical climate and environment.Secondly,we developed assay methods for Ascaris suum eggs development and the quantitative assay methods for Ascaris suum larvae migration in order to test anthelminthic efficacy and resistance,which may be used in anthelminthic resistance detection and screening of new antiparasitic drugs.Thirdly,we studied the effects of bovine bile and its main components on the egg hatching,and the viability of Ascaris suum larvae.The biochemical mechanism of the bioactive constituents in the bile that affect Ascaris egg hatching and larval migratory activity were preliminarily analyzed.To investigate bile effects on the Ascaris suum egg hatching and larva movement may help understand the host specificity of Ascaris suum and provide new approach in biological control of animal parasitic nematodes.The results are as follows:(1)From 2015 to 2018,a total of 19606 samples of pig intestines,faeces,soil and water were collected from 18 counties and cities in Hainan(all counties and cities in Hainan,excluding Sansha city),including 68 villages,18 slaughterhouses,64 small outdoor farms,95 small-scale intensive farms and 38 medium-scale intensive farms.The sampling sites cover the whole land area of Hainan Island.Results:the detection rates of Ascaris suum eggs in faeces samples from various counties and cities were 7.6%in the eastern Hainan,14.8%in the central part,and 13.8%in the western region.The detection rates of fecal samples were 2.5%in suckers,30.9%in weaners,30.7%in growers and 30.9%in sows.The detection rates of intestinal adult worms in slaughterhouses were 1.2%?3.6%,and the range of worm burdens was 1-8.The detection rates of eggs in soil samples were 7.2%in pastoral vegetable fields and 2.6%along rivers;the detection rates of eggs in water were 1.2%in rice field,0%in reservoir and 5%in rain water accumulation.The overall prevalence of Ascaris suum infection in the "Hainan black pig" is low,showing the infection is "regional endemic".Meanwhile,the epidemic factors of swine ascariasis are complex.The spread of Ascaris eggs varied greatly with differences in the dry and wet seasons of Hainan.(2)We have used an assay method for the determination of ovicidal activity of anti-helminthic drugs ex vivo and in vitro,and identified the ovicidal activity of flubendazole in this experiment,and found no ovicidal activity of avermectin,doramectin,and ivermectin.Results:Avermectin,doramectin and ivermectin did not show any ovicidal activity against Ascaris eggs compared with the control group(P>0.05).We observed the Ascaris eggs from pigs treated with fluorbendazole were stopped for development in early stages.The development of eggs was inhibited and most eggs remained at one cell stage(85.5%),only 6.3%of the eggs developed into larvae.Ascaris eggs treated with anthelmintics(doramectin,0.04 ppm and flubendazole,1.0 ppm)were used to infect mice artificially.The infectivity of Ascaris eggs was identified by observing the changes of the number of Ascaris larvae in the lungs.The results showed that there was no significant effect on the infectivity of Ascaris eggs treated with anthelminthics in vitro(P>0.05).The method can be used to detect the any drug components or screen plant extracts with ovicidal activity.(3)In this study we developed a combined multi-well culture and agar gel larval migration assay to test the effect of benzimidazole and tetrahydropyrimidin/imidazothiazole anthelmintics against nine isolates of A.suum.Drugs tested were thiabendazole,fenbendazole,mebendazole,levamisole,and pyrantel.The percentages of larvae that migrated to the surface of each treated and control well were used to calculate the drug concentration which inhibits 50%of the larvae migration(EC50).The values of EC50 of thiabendazole,fenbendazole,mebendazole,levamisole,and pyrantel against A.isolates ranged 74?150,4.9?13.9,2.3?4.3,358?1150 and 1100?4000 nM,respectively.This combined multi-well culture and agar gel larval migration assay was a sensitive bioassay for anthelmintic activity and could serve as an in vitro method to detect for lowered drug efficacy against A.suum or possibly to screen for anthelmintic drug candidates.(4)Seven bile acids,LCA,CA,DCA,CDCA,HC,GDCA and TCA in three different biles(of cattle,swine and chicken)were determined by HPLC.There were 6 bile acids in bovine bile,5 in swine bile and 1 in chicken bile,the highest content of cholic acid(CA)in bovine bile(16.08 g/mL)and the highest content of deoxycholic acid in swine bile(6.42 g/mL)were found.The content of geo deoxycholic acid(4.01 g/mL)in chicken bile was higher than that in bovine(0.15 g/mL)and swine(0.08 g/mL).The results showed that the larval migration rate was the highest by adding 5%bovine bile,with the number of 32.51%.With the increase of bile concentration from 5%,the number of larvae migrated gradually decreased.When bile concentration reached 80%,the larvae migration rate was the lowest(6.24%).In group 2(chicken bile),the effect of concentration change on larval migrationl rate was not obvious.Compared with the control well,the addition of chicken bile inhibited the migration behavior of Ascaris suum larvae.In the experimental group 3(swine bile),the addition of 5-10%swine bile was beneficial to larval migration.With the increase of the concentration of bile,the larval migration rate was similar to that of the experimental group 1,and gradually decreased.The effect of seven bile acids on the migration activity of Ascaris suum larvae:with the increase of the concentration of each test group,the larval migration rate did not change significantly compared with the control group,and the difference between the test and control groups was not significant(P>0.05).Single bile acid compound could not stimulate the migration activity of Ascari suum.In order to understand the selectivity of agar gel migration test to bile species,the physical properties of bile from cattle,swine and chicken were compared.It was found that the volume of ox bile was the largest,and that of swine bile was the strongest in stimulate the migration,and that of cattle,swine and chicken were partly soluble in water.The biles of three animals dissolved in anhydrous ethanol showed precipitation and turbidity.It was insoluble in acetone and soluble in DMSO.Overall,this study investigated and analyzed the prevalance of Ascarsi suum infection in varies farmes in Hainan,presence of Ascarsi suum ages and adutls,which may provide insight into new control program of Ascarris suum of the "Hainan black pig".Flubendazole showd the ovicidal activity.A preliminary biochemical analysis of the biles was done for seeking individual bile components for activating larval migration of Ascarsi suum.Although no active component was found in stimulating the larval movement,the progress in this study is a good start for the future identification of bile active component mediating larval migration of Ascarsi suum. |
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