Prevalence Of Four Intestinal Protozoa Carried By Non-biting Flies In Pig Farms In Some Areas Of China

Non-biting flies,such as Musca domestica and Chrysomypha megacephala,are closely related to human and animal life and can complete their entire life cycle in human habitats and animal feeding grounds.Non-biting flies can carry a variety of parasites,among which Cryptosporidium,Giardia duodenalis,Blastocystis sp.,and Enterocytozoon bieneusi are the most common intestinal pathogens in pigs,with a high infection rate,and are all zoonotic intestinal parasites.The purpose of this study was to investigate the prevalence of four intestinal protozoa species in non-biting flies from pig farms in some areas of China.Prevalence and genetic characteristics based on the 18 S r RNA gene of Cryptosporidium,bg,tpi and gdh genes of G.duodenalis,ITS gene of E.bienesusi,and SSU r RNA gene of Blastocystis sp.using PCR amplification.To determine the potential threat of non-biting flies as vectors for the transmission of parasitic diseases in pig farms.1.Prevalence of four intestinal protozoa carried by non-biting flies in pig farms in some areas of ChinaIn this study,3420 flies from pig farms were randomly collected from 16prefecture-level cities in 6 provinces.A total of five non-biting flies were identified by combining morphological identification and molecular identification by DNA barcoding,namely,M.domestica,Lucilia sericata,C.megacephala,Aldrichina grahami and Helicophagella melanura.The dominant fly species is M.domestica,accounting for 91.2%(3120/3420).Two sets of positive samples were successfully amplified based on the Cryptosporidium 18 S r RNA gene,and a total of two Cryptosporidium species,C.suis and C.scrofarum,were identified.A total of 8positive samples were amplified in non-biting flies based on the G.duodenalis bg gene,all of which were assemblage E.Further amplification was based on the gdh and tpi genes,and a total of 3 positive samples were amplified at the gdh gene,all of which were assemblage E.Two positive samples were amplified at the tpi gene,both of which were assemblage E.A total of 2 samples were amplified at all three genes at the same time,constituting only 1 MLG E1.A total of 24 groups of positive samples were amplified in non-biting flies based on the ITS gene of E.biensusi.After sequence alignment and splicing,a total of 2 genotypes were identified,namely Ebp C and Ebp A,all belonging to Group1,which has an important risk of zoonotic disease.Based on the SSU r RNA gene of Blastocystis sp.,a total of 107 positive samples were amplified in non-biting flies.After sequence alignment and splicing,a total of 3zoonotic gene subtypes were identified,namely ST3,ST5,and ST1.2.Molecular epidemiological investigation of four species of intestinal protozoa infecting pigs in some areas of ChinaAt the same time,the prevalence of Cryptosporidium,G.duodenalis,E.biensusi and Blastocystis sp.in 1794 fecal stool samples from the same farm where fly samples were collected was calculated.The results show that the prevalence of Cryptosporidium,G.duodenalis,E.biensusi and Blastocystis sp.were 2.3%(41/1794),2.1%(38/1794),12.6%(226/1794)and 30.8%(553/1794),respectively.By statistical analysis,the differences in the prevalence of all four intestinal protozoa infections in pigs in different regions were highly significant(P<0.01).The differences in the prevalence of Cryptosporidium,E.biensusi and Blastocystis sp.infection in different ages of pigs were highly significant(P<0.01),and the prevalence of G.duodenalis was significant(P<0.05).Based on the Cryptosporidium 18 S r RNA gene,two Cryptosporidium species,C.suis and C.scrofarum,were identified after sequence alignment and splicing.38 positive samples were amplified based on the bg gene of G.duodenalis,which were assemblage E(n=33)and assemblage A(n=5),and then further amplified based on the multiple sites of gdh and tpi genes.A total of 10 positive samples were amplified from the gdh gene.The sequencing results showed that they were assemblage E(n=7)and assemblage A(n=3),respectively.A total of 7positive samples were amplified from the tpi gene,and the sequencing results showed that they were assemblage E(n=4)assemblage A(n=3).A total of 7 samples were amplified at the same time in three genes,forming three MLG subtypes,namely MLG E1,MLG E2 and MLG A1.A total of 10 genotypes,Ebp C,Ebp A,Pig EBITS5,CHG19,HIJ-IV,Henan-IV,CHS5,O,BEB6 and H,nine genotypes belonging to Group1 and one genotype belonging to Group2,were identified after sequence alignment and splicing based on the ITS gene of E.biensusi.Based on the SSU r RNA gene of Blastocystis sp.,three subtypes of zoonotic genes were identified after sequence alignment and splicing,namely ST5,ST3,and ST1.3.The role of non-biting flies in the transmission of four intestinal protozoa species were analyzed based on genetic characteristicsIn this study,through homology analysis and phylogenetic analysis,C.suis was identified from the vector M.domestica and the host pig in the same place;C.scrofarum was identified from the vector H.melanura and the host pig in the same place;assemblage E was identified from the vector M.domestica and the host pig in the same place;the same zoonotic genotype Ebp C was identified in the vector M.domestica and the host pig in the same place;the subtypes ST1,ST5 and ST3 of the zoonotic genes of the same Blastocystis sp.were identified in the vector M.domestica,L.sericata and A.graham and the host pigs in the same place.In summary,this study investigated the carriage of four intestinal protozoa by non-biting flies in some pig farms in China,and also counted the prevalence of four intestinal protozoa in pig fecal samples from the same field where flies samples were collected.C.suis,C.scrofarum,assemblage E,Ebp C,ST1,ST5,and ST3 were identified from non-biting flies and pigs in the same place.The results show that non-biting flies have potential transmission risks,and this study provides a reference for the prevention and control of parasitic diseases in pig farms and public health.