Molecular Investigations Of Cryptosporidium Of Birds In Hubei Province,China

Cryptosporidium spp.are widespread and important zoonotic parasitic protozoa,which infect humans and other vertebrates.Species within this genus are transmitted via the faecal-oral route,often through direct contact with infected people or animals or the ingestion of contaminated food and/or water,resulting in severe diarrhoea in mammals,and posing a great threat to human health.Cryptosporidium meleagridis is the third major species causing human cryptosporidiosis in the world,and also an important pathogen infecting bird.The cross-transmissibility of C.meleagridis between birds and humans has been reported in Sweden and Peru.In addition,in a recent survey,we were surprised to find that C.meleagridis subtype IIIb was associated with diarrhoea in children in Wuhan city,Hubei Province.Hence,we conducted a molecular epidemiological investigation of Cryptosporidium of birds in this geographical region to explore possible sources of infection of C.meleagridis in children and to assess transmission risk of this zoonotic disease by birds.Between July and November 2017,471 fresh faecal samples were collected from chickens of different age groups from 14 medium-to large-sized farms in six distinct geographical regions(Huanggang,Suizhou,Wuhan,Xiantao,Xiangyang and Yichang)of Hubei Province.PCR-coupled sequencing analyses of regions in the small subunit(SSU)of the nuclear ribosomal RNA and 60 kDa glycoprotein(gp60)genes were utilised to characterise Cryptosporidium in faecal samples from chickens.Cryptosporidium baileyi(33/471;7.0%)and C.meleagridis(15/471;3.2%)were identified in chickens on eight farms in five of the six regions.The prevalence of C.meleagridis was significantly higher in younger(≤ 4 months)than in older chickens(> 4 months),and no significant age-associated difference in the prevalence of C.baileyi was recorded.For C.meleagridis,two subtype families,IIIb and IIIe,were defined;some of the subtypes(i.e.IIIbA26G1R1 b and IIIbA22G1R1c)identified matched those identified previously in diarrhoeic children in Wuhan.These findings suggest that C.meleagridis subtypes IIIbA26G1R1 b and IIIbA22G1R1 c are cross transmissible between chickens and humans,raising awareness about the significance of birds as possible reservoirs of zoonotic variants of Cryptosporidium.Subsequently,we undertook a molecular epidemiology study of Cryptosporidium of pet birds in Wuhan city.Between August and December 2018,a total of 322 fresh fecal samples from 44 species representing 6 orders of pet birds from 11 pet bird markets were collected.PCR-coupled sequencing of regions in SSU and gp60 genes was used to identify Cryptosporidium in the sample set.The overall prevalence of Cryptosporidium in pet birds was 20.2%(65/322),including C.baileyi(n = 29),Cryptosporidium galli(n = 3),C.meleagridis(n = 7),Cryptosporidium muris(n = 1),the Cryptosporidium avian genotype III(n = 12)and the avian genotype V(n = 13).Samples from 15 species of pet birds were test-positive,e.g.for myna,white java sparrow,gouldian finch,cockatiel,pigeon and ostrich.Interestingly,C.galli infection occurred only in Passeriformes,while avian genotype III/V appeared mostly in Psittaciformes.For C.meleagridis,a phylogenetic analysis revealed that only one subtype(IIIeA17G2R1)was defined,which neither matched those identified in farmed chickens nor in diarrhoeic children in Wuhan.Taken together,this is the first molecular study to report the genetic prevalence and identity of Cryptosporidium in farmed chickens and pet birds in Hubei Province.The findings suggest that birds serve as potential reservoirs of zoonotic variants of Cryptosporidium,emphasising the need for larger molecular epidemiological studies of Cryptosporidium,particularly C.meleagridis,in humans and birds in China and elsewhere using multiple informative genetic loci.Further studies are also needed to undertake temporal and spatial studies of the prevalence of Cryptosporidium species,genotypes and subtypes in a range of animals(e.g.,ruminants,carnivores and fish)in one of the fastest growing economic regions in China(Hubei).