Advances and Perspectives on the Epidemiology of Bovine Cryptosporidium in China in the Past 30 Years

Major progress has been made in understanding the epidemiology of bovine Cryptosporidium in China in the past 30 years. The overall infection rate in that period was 14.50% (5265/36316), with different prevalence being observed among dairy cattle, yaks, beef cattle, and buffalo. The infection rate declined as the animals’ ages increased and the lowest prevalence occurred in winter. Ten Cryptosporidium species and two genotypes have been found in cattle, with Cryptosporidium parvum, C. andersoni, C. bovis, and C. ryanae being the commonest species. Cryptosporidium bovis rather than C. parvum predominated in preweaned dairy cattle, and C. parvum IIdA15G1 and IIdA19G1 were the only subtypes detected in dairy cattle. Two subtype families, IIa and IId, were found in yaks. Population genetic analysis detected an epidemic population structure in C. andersoni, which suggested that the prevalence of C. andersoni in China is not attributable to the introduction of dairy cattle. Moreover, C. parvum IId subtypes probably dispersed from western Asia to other geographic regions based on population genetic analysis of isolates from China, Sweden, and Egypt. Therefore, we hypothesize that Cryptosporidium was introduced into China in the past, and different populations formed progressively in various hosts in response to diverse factors, including the transmission dynamics, geographic isolation, host specificity, and large-scale farming. More epidemiological studies are required to test this hypothesis and to clarify the prevalence and transmission of Cryptosporidium species in China.


INTRODUCTION
Cryptosporidium spp. are important zoonotic agents infecting a wide spectrum of vertebrate hosts (Xiao et al., 2004;Wang et al., 2011b). There is extensive genetic variation within the genus Cryptosporidium. To date, thirty-one recognized species and more than 60 Cryptosporidium genotypes have been discovered (Xiao et al., 2004;Wang et al., 2010;Ryan et al., 2014). Recent studies into the global causes of severe diarrhea in children suggested that Cryptosporidium is the second most important diarrheal pathogen after Rotavirus (Kotloff et al., 2013;Checkley et al., 2015;Vinayak et al., 2015).
Members of the genus Cryptosporidium complete all developmental stages in a single host. Sporulated oocysts, containing four sporozoites, are released from an infected host upon defecation (O'Hara and Chen, 2011). Following ingestion by a suitable host, the motile, infective sporozoites are released through a suture in the oocyst wall and parasitize epithelial cells of the gastrointestinal tract or other tissues (Reduker et al., 1985;O'Hara and Chen, 2011). In these cells, the parasites undergo asexual multiplication, which produces Type-I and Type-II merozoites (Current and Reese, 1986). Type-II merozoites ultimately produce either male or female equivalent sexual reproductive stages, microgametocytes and macrogametocytes, respectively (O'Hara and Chen, 2011). After the macrogamonts are fertilized by the microgametes, oocysts are formed and then sporulated in the infected host. Thick-walled and thin-walled representing two different types of oocysts are produced. The former is commonly excreted from the host, and the latter is primarily involved in autoinfection. Cryptosporidium can be transmitted by the fecal-oral route, via either direct contact or ingestion of contaminated food or water (Xiao et al., 2004;Wang et al., 2011c).
Cattle are the mammals in which Cryptosporidium infection is most commonly found, and preweaned calves are considered the most important reservoir for zoonotic infection (Wang et al., 2011b;Li F. et al., 2016). Cryptosporidium parvum, C. bovis, C. andersoni, and C. ryanae are predominantly responsible for bovine cryptosporidiosis, although several other Cryptosporidium species and genotypes are also discovered in cattle, including C. felis, C. hominis, C. suis, C. scrofarum, C. meleagridis, and C. suis-like genotype Wang et al., 2011b;Zhang et al., 2013;Huang et al., 2014;Robertson et al., 2014). Studies conducted in numerous industrialized nations have suggested that C. parvum is the species most often found in preweaned calves and that it is a significant cause of diarrhea (Wang et al., 2011b). Cryptosporidium bovis and C. ryanae usually infect weaned calves and yearlings, although C. bovis is more commonly seen than C. ryanae, but neither are associated with the occurrence of diarrhea Wang et al., 2011b). In contrast, C. andersoni is commonly observed in adult cattle and has been associated with gastritis, reduced milk yield, and poor weight gain (Esteban and Anderson, 1995;Wang et al., 2011b).
Cryptosporidium infections are frequently detected in humans and various domestic and wild animals in China (Wang et al., 2008(Wang et al., , 2010(Wang et al., , 2011c(Wang et al., , 2014aFeng et al., 2012). Among these, cattle are one of the major targets in which Cryptosporidium is studied. To date, 97 papers involving 24 provinces, autonomous districts, and municipalities of China have been published on Cryptosporidium infections in cattle since the first case was reported in Zhou et al. (1985). The present paper focuses on the advances in the molecular epidemiology of bovine Cryptosporidium that have occurred in China in the past 30 years.

Cryptosporidium SPECIES DISTRIBUTION
A total of 1690 Cryptosporidium-positive isolates were genotyped and ten Cryptosporidium species and two genotypes were identified, including C. bovis, C. andersoni, C. ryanae, C. parvum, C. xiaoi, C. ubiquitum, C. meleagridis, C. hominis, C. tyzzeri, C. serpentis, C. suis-like genotype, and a new genotype ( Table 1). Cryptosporidium bovis (129/364) rather than C. parvum (119/364) was the predominant Cryptosporidium species in preweaned dairy cattle (Wang et al., 2011b;Zhang et al., 2013;Cui et al., 2014;Huang et al., 2014;Qi et al., 2015). The earliest detection of C. bovis was in 1-week-old calves, indicating that the prepatent period was shorter than the previously recorded 10-12 days (Wang et al., 2011b). Cryptosporidium andersoni was most commonly found in heifers and adult cattle (Wang et al., 2011a). In contrast, another two common species, C. bovis and C. ryanae, occurred at different rates in different epidemiology studies. It is noteworthy that C. tyzzeri (formerly Cryptosporidium mouse genotype I) and C. serpentis probably arose from contamination (Chen and Huang, 2012), and the authors stated in the GenBank submissions that identical sequences (DQ855266 and DQ855267) were found in isolates from pigs with a reverse transcription-PCR analysis of the small subunit ribosomal RNA (Chen and Huang, 2007), and several SSU rRNA gene sequences of C. tyzzeri (EU369382, EF025503, EU369384, EU369381, and EU369383) previously isolated from bovine samples have also been deposited in GenBank. Neither C. tyzzeri nor C. serpentis is a known bovine parasite (Wang et al., 2011a,b).
As in dairy cattle, C. parvum, C. andersoni, C. bovis, and C. ryanae were also the four Cryptosporidium species most commonly identified in yaks, although several other Cryptosporidium species and genotypes have been detected (Table 1). However, the distribution of different Cryptosporidium species according to yak age is still unclear. A study conducted in Qinghai suggested that C. bovis was the predominant species in yaks ≤ 2 years old, whereas C. parvum was more common in older yaks (Mi et al., 2013). In contrast, a recent study in Tibet showed that C. andersoni was predominant in 1-2-yearold yaks, whereas C. bovis was commonly found in older yaks (Li P. et al., 2016). Therefore, more studies are required to clarify the Cryptosporidium distributions according to age in different groups of animals. Two studies that genotyped Cryptosporidiumpositive isolates from beef cattle and buffalo identified only C. andersoni, C. bovis, and C. ryanae (Ma et al., 2015;Qi et al., 2016).

Cryptosporidium SUBTYPES
Subtyping tools have been used extensively in studies of the transmission of C. hominis, C. parvum, and several other related Cryptosporidium species, including C. meleagridis and C. ubiquitum, in both humans and animals (Xiao, 2010;Li et al., 2014;Ryan et al., 2014). One of the most frequently used subtyping tools is a DNA sequence analysis of the 60-kDa glycoprotein (gp60, also known as gp40/15) gene (Ryan et al., 2014).

POPULATION GENETICS
With the development of Cryptosporidium subtyping tools, it has become possible to assess the genetic and population structures of Cryptosporidium species (Xiao and Ryan, 2008). A total of 149 C. andersoni isolates from beef cattle (n = 38) and dairy cattle (n = 111) were subtyped with a multilocus sequence typing (MLST) tool based on the MS1, MS2, MS3, and MS16 loci. Fourteen MLST subtypes were identified and A4,A4,A4,A1 was the predominant subtype Zhao et al., 2013;Qi et al., 2016). To test the possibility that this linkage disequilibrium (LD) was attributable to the clonal expansion of one or more subtype, masking the underlying equilibrium, an LD analysis was conducted using only the MLST subtypes (by considering each group of isolates with the same MLST subtype as one individual , 14 MLST subtypes were used in this analysis), with LIAN version 3.7. This analysis suggested that the C. andersoni population in cattle in China had an epidemic population structure (I S A = 0.0010, V D < L). An analysis using STRUCTURE 2.3.4 with K-means partitional clustering and the admixture model revealed three ancient lineages among the 149 C. andersoni specimens (Figure 1).
Cryptosporidium parvum is another species that has been targeted for the population genetic analysis in cattle. Cryptosporidium parvum IId isolates (n = 111) from several species of animals in China, Sweden, and Egypt (Wang et al., 2014b) were subtyped with an MLST tool based on 12 microsatellite, minisatellite, and single-nucleotide polymorphism loci (Wang et al., 2014b). Host adaptation and significant geographic segregation were both observed in the MLST subtypes. A clonal population structure was detected in the C. parvum IId isolates from China and Sweden. Three ancestral lineages and the same RPGR (retinitis pigmentosa GTPase regulator) sequence were shared by the isolates examined (Wang et al., 2014b). The authors concluded that the C. parvum IId subtypes probably dispersed from western Asia to other geographic regions (Wang et al., 2014b;Zahedi et al., 2016).

CONCLUSION AND PERSPECTIVES
Epidemiological data suggest that Cryptosporidium infections are commonly found in cattle in China. The infection rate declines as the age of the animals increases, and the lowest infection rate occurs in winter. Similar to the species distributions reported in other countries and areas of the world, C. parvum, C. bovis, C. andersoni and C. ryanae are the four commonest Cryptosporidium species in cattle. Cryptosporidium bovis rather than C. parvum was the dominant Cryptosporidium species in preweaned dairy cattle, which differs from the dominant species in this age group in other countries and areas of the world. Uniquely, the C. parvum subtypes identified in dairy cattle were all zoonotic IIdA15G1 or IIdA19G1 (Wang et al., 2014b).
Population genetic analyses of 149 C. andersoni isolates in three published studies confirmed an epidemic population structure, and as proposed in a previous study, these data suggest that the prevalence of C. andersoni in China is not attributable to the introduction of dairy cattle . According to a population genetic analysis, the C. parvum IId subtypes probably dispersed from western Asia to other geographic regions (Wang et al., 2014b). Therefore, we hypothesize that Cryptosporidium was introduced into China at some time in the past, and then different Cryptosporidium populations developed progressively in various hosts in response to diverse factors, including the transmission dynamics, geographic isolation, host specificity, and large-scale farming. More epidemiological studies are required to confirm this hypothesis and to clarify the transmission and public-health impact of Cryptosporidium species in China.

AUTHOR CONTRIBUTIONS
LZ had the ideal for the review and revised the manuscript. RW wrote the paper. RW, GZ, and YG reviewed and abstracted data from each selected article.

FUNDING
This study was supported in part by the Key Program of the National Natural Science Foundation of China (31330079), the National Natural Science Foundation of China (31672548, 31302079, and 31572509), the Program for Science and Technology Innovation Talents in Universities of Henan Province (16HASTIT018), and the Natural Science Foundation of Henan Province (162300410129).