| Cryptosporidium spp.,Enterocytozoon bieneusi,Giardia duodenalis and Blastocystis sp.are important zoonotic intestinal protozoa,which infect a wide range of hosts,including humans,mammals,birds and reptiles.The main route of transmission of the four kinds of intestinal protozoa is fecal-oral transmission,which is prevalent all over the world.Human infection with intestinal protozoa will lead to acute or chronic diarrhea,nausea,vomiting and other clinical symptoms.Children infected with Cryptosporidium and Giardia duodenalis will lead to growth retardation and cognitive impairment.Cattle are important hosts of four kinds of intestinal protozoa,and the zoonotic species and genotypes they carry can be used as a source of infection to threaten the health of human beings and other animals,leaving hidden dangers to public health and safety.In order to understand the prevalence and genotype distribution of four kinds of intestinal protozoa in Longjiang Wagyu cattle in Heilongjiang province,1003 fecal samples of Longjiang Wagyu cattle were collected from Longjiang Wagyu cattle farms in Heilongjiang province,and the molecular epidemiological investigation of four kinds of intestinal protozoa was carried out,and the risk of zoonosis was evaluated.Four kinds of intestinal protozoa were detected by nest PCR based on different gene loci.The positive results were bidirectional sequencing and sequence splicing,then BLAST alignment and phylogenetic analysis were carried out,and the following results were obtained:1.Based on the small subunit ribosomal RNA gene of Cryptosporidium,the total infection rate of Cryptosporidium was 1.5 %(15/1003).The infection rate of young cattle was the highest,which was 2.7 %.The infection rates of pre-weaning calves and post-weaning calves were 2.1 %and 1.0 %,respectively.No Cryptosporidium infection was detected in breeding cows.Among different areas,the infection rate in Fuyu is the highest,which is 3.1 %,and that in Longjiang is the lowest,which is 1.0 %.The infection rate in Zhaoyuan and Durbert is 1.2 % and 1.4 %,respectively.Statistical analysis showed that the Cryptosporidium infection rate in Longjiang Wagyu cattle was not significantly different in different regions and different ages(P >0.05).Three species of Cryptosporidium were found in this study,namely,(C.andersoni,n=6),(C.muris,n=4)and(C.parvum,n=5).All three species of Cryptosporidium were detected in human body.There was an age correlation between C.andersoni and C.parvum,that is,C.parvum was only found in pre-weaned calves and C.andersoni was only detected in young cattle.Based on the gp60 locus,the gene subtype of C.parvum was identified as zoonosis subtype IId A20G1.2.Based on the gene of ribosomal transcribed spacer region of Enterocytozoon bieneusi,the total infection rate of Enterocytozoon bieneusi was 1.7 %(17/1003).The infection rate of preweaning calves was the highest,which was 2.1 %.The infection rate of young cattle was the lowest,which was 1.2 %.The infection rates of post-weaning calves and breeding cows were1.6 % and 2.0 %,respectively.Among different areas,the infection rate in Fuyu is the highest,which is 2.5 %,while that in Longjiang is the lowest,which is 1.3 %.The infection rate in Zhaoyuan and Durbert is 1.8 % and 1.7 %,respectively.Statistical analysis showed that the Enterocytozoon bieneusi infection rate in Longjiang Wagyu cattle was not significantly different in different regions and different ages(P > 0.05).In this study,four genotypes of Enterocytozoon bieneusi were identified as BEB4(n=2),BEB6(n=5),I(n=3)and J(n=7).Phylogenetic analysis showed that these four genotypes were all located in Group2,which was host-specific and mainly infected livestock.BEB4,BEB6,I and J found in this study has also been found in humans.17 Enterocytozoon bieneusi positive samples were amplified by nest PCR based on 4 loci,and multi-site sequence analysis(multilocus sequence typing,MLST)was performed.7,8,3 and 7 bands were amplified at MS1,MS3,MS4 and MS7 loci,respectively.The ITS genotypes were all J genotypes.In this study,only 6 samples with 3 or more loci were analyzed by sequence alignment.The results showed that there were 3,3,1 and 3 subtypes at 4loci,respectively,forming 6 multi-locus genotypes(Multilocus Genotypes,MLGs),which showed that genotype J had rich genetic diversity.3.Nested PCR amplification based on the β-giardia(BG)gene of Giardia duodenalis showed that the total infection rate of Giardia duodenalis was 1.1 %(11/1003),in which the infection rate of young cattle was the highest(2.7 %),that post-weaning calves was 1.3 %,and no G.duodenalis infection was detected in pre-weaning calves and breeding cows.Among the sampling areas,the infection rate of Longjiang was the highest(1.8 %),that of Zhaoyuan and Durbert was 0.6 % and 1.0 %,respectively,and that of Fuyu was 0 %.Statistical analysis showed that the G.duodenalis infection rate in Longjiang Wagyu cattle was not significantly different in different regions and different ages(P >0.05).In the present study,two genotypes were identified,which were Assemblage A(n=1)and Assemblage E(n=10).4.Nest PCR amplification based on Blastocystis SSU r RNA gene showed that the total infection rate of Blastocystis was 2.1 %(21/1003).The infection rate of Blastocystis was the highest in young cattle(3.9 %).The infection rates of pre-weaning calves and post-weaning calves were 2.1 % and 1.9 %,respectively.There was no Blastocystis infection in breeding cows.Among different areas,the infection rate in Zhaoyuan is the highest,which is 3.0 %,and that in Longjiang is the lowest,which is 1.3 %.The infection rate in Fuyu and Durbert is 2.5 % and2.4 %,respectively.Statistical analysis showed that the Blastocystis infection rate in Longjiang Wagyu cattle was not significantly different in different regions and different ages(P >0.05).In this study,three subtypes of Blastocystis were identified,which were ST10(n=11),ST12(n=7),and ST14(n=3).In the present study,molecular biological methods were used to investigate the infection and the distribution of species and genotypes of four kinds of intestinal protozoa in Longjiang Wagyu cattle in Heilongjiang province.The results showed that the total infection rate of intestinal protozoa in Longjiang Wagyu cattle in Heilongjiang province was 6.4 %(64/1003),and the dominant species was Blastocystis(2.1 %,21/1003).In this study,ST12 subtype was detected in Heilongjiang cattle for the first time.There are 9 species or genotypes that can infect humans,which is of public health significance.The results provide a scientific basis for the prevention and control of four kinds of intestinal protozoa in Heilongjiang province. |
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