Epidemiology Investigation On Intestinal Protozoa And Species Identification And Genotyping Of Cryptosporidium Isolates From Some Wild Animals

Cranes,especially chicks,may succumb to acute infections resulting in hepatitis, bronchopneumonia,myocarditis,splenitis,and enteritis.Cranes may also develop chronic, subclinical infections characterized by granulomatous nodules in various organs and tissues.34 faecal specimens of captive adult cranes representing 5 species,collected in Zhengzhou Zoological Park,were examined.The results showed,the infection ratio of Coccidia was 35.29%, the average infection intensity(OPG)was 2.08×10~4.3 species of Coccidia in cranes were discovered,namely,Eimeria gruis,E.eichenowi and lsospora lacazei.Coccidia of minks mainly invade and reside in the liver and intestinal tract,cause clinical signs such as diarrhea and lead to mink death.To determine the prevalence and species of coccidian oocysts,469 faecal specimens collected in Suning county in Hebei Province were examined.The results showed that,the infection rate of Coccidia was 34.33%(161/469).Thereinto,the infection rate of fitchet was 34.33%(80/233),the gray sable was 56.25%(27/48)and the sable was 28.72%(54/188).Seven Coccidia spp.in minks were discovered,namely,Eimeria hiepe,Isospora eversmann,1.Iaidlawi,E.furonis,E.icidea,unnamed E.sp and a single sporangium Coccidia.Cryptosporidiosis is a zoonotic protozoan disease of worldwide distribution,affecting a wide range of vertebrate hosts.In animals,Cryptosporidium spp.mainly cause digestive and respiratory system diseases,leading to poor health or death,and significant economic losses.In immunocompetent humans,Cryptosporidium causes acute infections of the digestive system, while in irnmunocompromised patients,especially AIDS patients,it causes a chronic, life-threatening disease.There are many reports on Cryptosporidium from wild animals,and 10 of 16 Cryptosporidium valid species have been found in wild animals so far.In that,6 species can infect humans,i.e.,C. muris,C..parvum,C.canis,C.felis,C.meleagridis and C.hominis.Therefore,there is most important public health significance to study the cryptosporidiosis in wild animalsThere is no report on Cryptosporidium from wild animals in China so far.Based on it,we performed a survey on Cryptosporidium in wild animals,and identified the isolates by morphology,cross-transmission studies,and sequence analyses at 18S rRNA,HSP70,actin and COWP loci.Epidemiology survey were performed by Sheather's sugar flotation technique,and the objects studied included reptiles,camel,fox,precious birds,minks and other wild animals. Cryptosporidium was found in a bactrian camel from the wild animals rescue center of Henan Province,and minks from a farm in Suning county in Hebei Province.Survey results showed the mean prevalence of Cryptosporidium in minks was 1.71%(8/469). The infection rate for minks aged 5-6 months old was 2.17%(8/369),and 0(0/100)for 2-5 years old.The infection rate for female and male minks was 2.83%(6/212)and 1.27%(2/157), respectively.The partial 18S rRNA,70 kDa heat shock protein(HSP70)and actin genes of six isolates were amplified and sequenced.Sequence data were analyzed together with known Cryptosporidium spp.and genotypes.Results of this multi-locus genetic characterization indicated that the six Cryptosporidium isolates in this study shared the same sequences of the genes studied and were different from known Cryptosporidium species and genotypes.The closest relative was Cryptosporidium ferret genotype with seven,22,and two nucleotide differences in the 18S rRNA, HSP70 and actin genes,respectively.The homology to ferret genotype at the 18S rRNA locus was 99.1%,which is comparable to that between C.parvum and C.hominis(99.2%),or between C. muris and C.andersoni(99.4%).Therefore,the Cryptosporidium in minks in this study is considered a new genotype,the Cryptosporidium mink genotype.At the same time, cross-transmission studies showed Cryptosporidium mink genotype isolates did not infect Kunming mice.We analysed the sequences of two Cryptosporidium isolates from a bactrian camel.The results showed that the sequences of the 18S rRNA and COWP were identical to all other C.andersoni isolates although minor differences were noticed between the isolates and the USA isolate at the actin locus(99.2%of similarity).The sequence of the HSP70 was identical to the Japanese C. andersoni isolate,with minor difference from the Australian C.andersoni isolate(99.7%of similarity).Cross-transmission studies demonstrated that the C.andersoni isolates did not infect immunosuppressed or immunocompetent Kun-ming mice,severe combined immunodeficiency mice and immunosuppressed or immunocompetent calves.Among the C.andersoni isolates reported so far,only isolates from Japan could infect SCID mice.Thus,the C.andersoni isolates from the bactrian camel were biologically similar to most bovine C.andersoni isolates characterized so far,but different from bovine isolates from Japan. This is the first report of cryptosporidiosis in wild animals in China.Especially,the discovery of Cryptosporidium mink genotype provided an essential complement for studying the evolution relationship among Cryptosporidium spp.,and got an important data for further studying the biological characteristics of Cryptosporidium mink genotype.