Molecular Epidemiology Of Cryptosporidium In Partial Areas Of China

Cryptosporidiosis is an recently emerging zoonotic infectious disease, which has been found in human cases in 106 countries and it is an important factor for acute diarrhea or chronic lethal diarrhea. Cryptosporidium spp. can infect more than 260 animals, including humans. It can be transmitted by the fecal-oral route, via either direct contact or ingestion of contaminated food or water. There is extensive genetic variation within the genus Cryptosporidium, in addition to 23 recognized species of Cryptosporidium, 74 Cryptosporidium genotypes with no designated species names have been described. Currently, no effective drug or vaccine is available for cryptosporidiosis. Thus, this disease seriously affects the growth and development of animals and human health, causing huge economic losses. In 1993, the outbreak of cryptosporidiosis occurred in Miwakkeii city in the United States has led to infections of 400,000 persons and more than 100 individuals were died, with the direct economic losses amounting to $ 96,200,000. Researches on the prevalence of Cryptosporidium spp. and the distribution of Cryptosporidium species/genotype/subtypes have important implications in understanding the spread of Cryptosporidium and the prevention and control of cryptosporidiosis. In the present study, molecular epidemiology study of Cryptosporidium was performed in six provinces (Henan, Sichuan, Shanxi, Guangxi, Heilongjiang, and Jilin) at four levels, i.e., epidemiological survey, species/genotype identification, subtype typing, and population genetic structure analysis.1. Sheather’s sugar flotation technique and modified acid-fast stain method were used to detect the Cryptosporidium oocysts in faeces of dairy cattle in epidemiological survey.Microscopic examination showed that the overall infection rate of Cryptosporidium spp. was 13.0% (276/2116) in dairy cattle in Henan province. The infection rate of Cryptosporidium spp. was 21.5% (172/801), 11.3% (86/758), 5.7% (15/262), and 1.0% (3/295) in pre-weaned, 3-11-month-old, 12-24-month-old, and more than 24-month-old dairy cattle, respectively (ρ< 0.01). The highest infection rate (50%) of Cryptosporidium spp. in pre-weaned calves was seen in summer and the lowest (17.3%) in winter (ρ> 0.05).2. Cryptosporidium samples were genotyped by restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the SSU rRNA gene, with the restriction enzyme SspI, VspI, and MboII being included.A total of 644 Cryptosporidium-positive samples from animals (dairy cattle, pigs, chichens, ducks, and ostriches) and humans were successfully genotyped and eleven Cryptosporidium species/genotypes were identified, including C. parvum (n=54), C. bovis (n=85), C. ryanae (n=19), C. andersoni (n=96), C. suis (n=94), pig genotype II (n= 14), C. baileyi (n=237), C. meleagridis (n=3), C. muris (n=10), C. hominis (n=9), C. felis (n=1), and Cryptosporidium mixed infections in calves (n=22). Among which, the C. parvum and C. andersoni in dairy cattle, the C. muris and C. meleagridis in birds, the C. suis and pig genotype II in pigs are zoonotic species/genotypes, having the risks of zoonotic transmission by animal-human route. That C. hominis and C. felis were detected in humans indicated that there existed two types of transmission route by human-human and animal-human in the areas studied.In pre-weaned calves, Cryptosporidium bovis was the most commonly identified Cryptosporidium and was found in all weekly age groups; C. parvum was the second most common species and was detected in seven age groups; the ratio of C. bovis and C. parvum were respectively 37.8% and 31.4% among all the Cryptosporidium-positive samples detected in pre-weaned calves. In contrast, C. andersoni was the most common Cryptosporidium species in post-weaned calves (3-11-month-old), heifers (12-24-month-old), and adult dairy cattle (more than 24 months of age), whereas C. bovis was only seen in 3 to 11-month-old calves. Cryptosporidium suis was the predominant Cryptosporidium species in pigs; except for 3 to 6-month-old pigs, the remaining age groups were all positive for two Cryptosporidium species/genotypes (C. suis and Cryptosporidium pig genotype II); C. suis was more often seen in younger pigs whereas the Cryptosporidium pig genotype II was relatively often seen in more than 6-month-old pigs. C. baileyi was the predominant species of Cryptosporidium detected in birds and was found in all age groups of chickens and 11 to 50-day-old ducks; in contrast, C. meleagridis were only identified in 31 to 120-day-old layer chickens. In ostriches, C. baileyi was found in lower than 1-year-old ostriches whereas C. muris was detected in more than 1-year-old ostriches. C. hominis was the predominant Cryptosporidium species in humans, mainly coming from infants/children under six years of age.Seasonal variation of Cryptosporidium species in pre-weaned calves showed that except for spring, the distribution of Cryptosporidium spp. differed significantly among seasons, with C. parvum dominating in summer and C. bovis in autumn and winter (ρ< 0.01).3. Subtyping of C. parvum and C. hominis was done by DNA sequence analysis of the 60 kDa glucoprotein (gp60) gene.In pre-weaned calves, sequencing analysis of the gp60 gene showed that 67 C. parvum samples all belonged to subtype IIdA19G1. In contrast, three subtype families were identified in the nine C. hominis samples: Ia (n=1), Ib (n=6), and Id (n=2). Among subtype family Ib, three subtypes were found: IbA16G2 (n=1), IbA19G2 (n=2), and IbA20G2 (n=3). Only one subtype each was present in subtype families Ia and Id: IaA9R3 (n=1) and IdA21 (n=2).4. Population genetic structure analysis of C. muris and C. andersoni was done by multilocus sequence typing (MLST) technique based on minisatellite sequences of MS1, MS2, MS3, and MS16 genes, and statistical analysis of the MLST data obtained by bioinformatics software.Fifty two C. muris and C. andersoni samples from different animals in Henan, Sichuan, Shanxi, Guangxi, Heilongjiang, and Jilin were typed by MLST technique. In total, nine C. muris isolates and 42 C. andersoni samples were successfully typed at all four genetic loci. Seven new subtypes were found and consisted of two C. muris and 10 C. andersoni MLST subtypes. Among which, the MLST subtype (4-4-4-1) was the predominant subtype of C. andersoni in cattle. The MLST subtypes of C. muris and C. andersoni differed in different animals (mice, ostriches, cattle, sheep, and camel), and there existed geographic distribution characterization. Population genetic analysis showed that cattle-derived C. andersoni in China was epidemic population structure.In summary, in this study, systematic epidemiological investigation of Cryptosporidium was performed in dairy cattle in Henan province; analyzing the distribution of Cryptosporidium species/genotype/subtypes in major livestock, poultry, and humans in Henan province; studying the population genetic structure of C. muris and C. andersoni in partial areas of China. Thus, the results have both academic value and important public health significance. In addition, the results also provided useful references for further studying the molecular epidemiology of Cryptosporidium in different areas, and for the effective prevention and control of cryptosporidiosis in China.