| The parasitic disease of sheep seriously restricts the development of sheep industry.Low return on feed,reduced milk production and meat production.Severe cases can lead to premature delivery of pregnant women,abortion,some cases of sheep deaths will occur,the direct economic income decline.In this study,577 samples were collected from the sheep farms in Tianjin area in.The parasitic eggs in feces were detected by various methods,and the status of infected nematodes in sheep in Tianjin area was investigated.In the meantime,Babesiidae found suspected worm like protozoa in the blood of the sheep,thus establishes the PCR detection method,and the application of the new method to detect 50 sheep blood samples,compared with the conventional Giemsa staining,after sequence alignment,the homology was 98%.1.Epidemiological investigation of intestinal nematodes in sheep in Tianjin.From March 2016 to January 2017,577 sheep feces were detected by parasites in Tianjin,including 3 large sheep farms and 2 small sheep farms.The detection methods include:saturated sodium chloride floating egg method,washing precipitation method,iodine staining method and saturated sucrose floating egg method.Results: the detection of nematode eggs,Tianjin local sheep infection rate was 6.97%.The lamb's infection rate was 12.86%,the growth of sheep was 6.74%,0.81% for fattening sheep.The infection rate was significantly higher than the growth of lamb and sheep sheep,the infection rate is greater than the growth of fattening sheep.Imported from Inner Mongolia sheep infection rate(78.26%)is far greater than the local Tianjin sheep infection rate(6.97%);the infection rate in autumn(10.23%)the average infection rate than those in other seasons(8.99%);small sheep infection rate(19.20%)than the large sheep infection rate(3.20%).Conclusion: the situation of sheep nematode infection in Tianjin area can not be ignored,so we should actively prevent and pay attention to the monitoring of parasitic infection.2.Establishment and optimization of PCR method for sheep BabesiidaeThis experiment through the staining results,selected molecular detection of nucleic acid detection samples to establish sheep trypanosomiasisevansi gradient method,and set the annealing temperature and number of cycles to optimize the temperature and cycle timesselected amplicons were specific,high yield and stable as the optimization result of PCR detection method.The results showed that the best reaction system of PCR reaction was 94 min pre denaturation at a temperature of about 94 DEG C,and it was modified at a temperature of about 1 min at a temperature of about 56 DEG C for about 72 DEG C at a temperature of 90 s for a period of up to 1 min,with a total circulation of about 39 times for a period of up to 72 DEG C,extending at a temperature of about 10 min for a temperature of 4DEG C.The PCR method established in this study has good specificity.3.application of PCR method for newly established BabesiidaeThe application of the new Babesiidae PCR,Morse worm detection method to detect the50 sheep blood,compare and select two sequences were sequenced with conventional Giemsa staining.The results showed that: in 50 blood samples,the positive samples detected by PCR is 13,the positive rate was 26%(13/50)positive samples detected,Giemsa staining is 8,the positive rate was 16%(8/50).PCR method to detect positive samples,and Giemsa staining positive samples were detected with the rate of 100%.Using NCBI sequence alignment on BLAST,the homology between the sequence and the reference sequence of GenBank: 98%(CP011906.1).The PCR detected the blood of the sheep for the Babesiidae worm. |
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