Establishment Of A Multiplex PCR And Indirect Immunofluorescence Assay For Cryptosporidium In Cattle

Cryptosporidiosis is a human-animal parasitic disease transmitted through water and food by protozoa of the genus Cryptosporidium.More than 40 species of Cryptosporidium have been identified so far,among which the main ones infecting cattle are Cryptosporidium parvum(C.parvum),Cryptosporidium bovis(C.bovis),Cryptosporidium andersoni(C.andersoni)and Cryptosporidium ryanae(C.ryanae).C.parvum is a human-animal pathogen that can be transmitted between humans and animals via the fecal-oral digestive and respiratory routes.Cryptosporidium infections can cause watery diarrhea,loss of appetite,lethargy and dehydration,and in severe cases can even cause death in humans and animals.Diarrheal symptoms usually appear about 3 to 4 days after oocyst ingestion and last about 1 to 2 weeks.There is no specific drug used to treat this disease in humans and animals,and early detection can effectively prevent the disease.Therefore,this study intends to establish a rapid,accurate and effective detection method for Cryptosporidium so as to effectively prevent and control the spread of Cryptosporidiosis,reduce its health hazards to humans and livestock,and promote the production of livestock.To establish a detection method for Cryptosporidium,it is necessary to first obtain its positive samples.Positive samples for Cryptosporidium were screened by nested PCR and DNA sequencing.Based on this,the gold standard of Cryptosporidium detection,the modified antacid stain,was used to directly detect Cryptosporidium oocysts to further identify positive Cryptosporidium infections for the sake of rigor of the test results.One of the four common species of Cryptosporidium in cattle is a human-animal species,so the enzymatic digestion method revealed that the human-animal C.parvum accounted for 8% of the sample.Positive samples for Cryptosporidium were obtained by the above steps.There are many pathogens,including Cryptosporidium,that cause diarrhea in calves,and clinical signs cannot accurately diagnose the type of pathogen that infects them.In this study,Multiplex Polymerase Chain Reaction was established for the three common pathogens causing calf diarrhea(Cryptosporidium,Giardia and Salmonella)selected.Specific primers were designed based on the conserved gene sequences(SSU r RNA,γ-giardin and Ivn A)of Cryptosporidium,Giardia and Salmonella published in NCBI,and the target bands of 803,519 and 1194 bp were expected to be amplified,respectively.Optimization of primer concentrations and annealing temperatures was performed,and the optimal concentrations of primers for Cryptosporidium,Giardia and Salmonella were determined to be 10,8 and 5 μmol/L,respectively,and annealing temperatures between 53 and 59°C resulted in the amplification of three bands of the correct size and specificity,which were determined to be the optimal amplification concentrations for multiplex PCR.Subsequently,the specificity of the method was verified by selecting several common diarrheal pathogens in calves,and the sensitivity of the method was tested by multiplicative dilution of the DNA concentration,and subsequent tests demonstrated the reproducibility of the method.A multiplex PCR method was developed and optimized for the rapid and accurate detection of three common diarrheal pathogens in calves,Cryptosporidium,Giardia and Salmonella,by the above method.The oocysts of Cryptosporidium are small,with a diameter of only 4-6 μm,and are easily missed by microscopic examination with the traditional staining method.In this study,we obtained P 786,a conserved protein on the wall of Cryptosporidium oocysts,prepared polyclonal antibodies and established an indirect immunofluorescence detection method,which can effectively detect Cryptosporidium in infected cattle.The established indirect immunofluorescence method was also tested for specificity and reproducibility,and the results showed that the established method has good specificity and reproducibility.In this study,an accurate and sensitive indirect immunofluorescence method was established to identify bovine Cryptosporidiosis using a conserved protein(P 786)on the oocyst wall of Cryptosporidium.The two established methods as well as the classical nested PCR were used to test 100 cattle manure samples from some areas of Taian City and compare the detection rates,which showed that the detection rates of Cryptosporidium by nested PCR,multiplex PCR and indirect immunofluorescence were 28,24 and 18%,respectively.The difference between the two established methods and the detection rate of nested PCR was small,which proved the good detection rate of the two established methods.In summary,two rapid,accurate and effective detection methods were established in this study.The first is a multiplex PCR that can rapidly and accurately detect three common diarrheal pathogens on calves,Cryptosporidium,Giardia and Salmonella;the second is an accurate and sensitive indirect immunofluorescence method to identify cryptosporidiosis in cattle using a conserved protein(P 786)on the oocyst wall of Cryptosporidium.This test can provide a rapid,accurate and effective identification method for practical production,and provide technical and temporal support for the development of effective epidemic prevention measures to better protect human and animal health and livestock production.