Cryptosporidium parvum GP60 subtypes present in diarrheic dairy calves of two biogeographical regions of Chile

Background Cryptosporidium is an apicomplexan zoonotic pathogen primary causing diarrhea in vertebrate hosts notably bovines and humans. Here, we characterized Cryptosporidium isolates by using the GP60 gene fragment of C. parvum to observe the dynamics of cryptosporidiosis transmission in dairy calves from two distant biogeographical regions of Chile (Metropolitan and Los Rios Regions). We collected 72 fecal samples from diarrheic calves screening the parasite carried out microscopy of an acid-fast staining smear and molecular characterization employing PCR to directly detect the Sanger GP60 C. parvum subtype and simultaneously in one selected sample the NGS prole of the GP60 same gene fragment to determine same and/or others Cryptosporidium subtypes Results The IIaA15G2R1 subtype was present in the 100% of the bovine fecal samples studied from Los Rios Region. Along with this same subtype, another two were observed in the Metropolitan Region, IIaA17G2R1 and IIaA17G4R1. The NGS analysis of a single selected GP60 PCR amplicon of one selected sample of our study showed similarly the Sanger sequencing determined subtype, the IIaA17G4R1 in 90% of readable sequences observed. By using this approach another multiple low frequency IIa subtypes of C. parvum were observed conrming that in an infected host multiple subtypes of the parasite can be present.

IIaA17G2R1 and IIaA17G4R1. The NGS analysis of a single selected GP60 PCR amplicon of one selected sample of our study showed similarly the Sanger sequencing determined subtype, the IIaA17G4R1 in 90% of readable sequences observed. By using this approach another multiple low frequency IIa subtypes of C. parvum were observed con rming that in an infected host multiple subtypes of the parasite can be present.
Conclusions Cryptosporidiosis in these dairy farms calves in Chile is produced by C. parvum limited number of subtypes, being IIaA15G2R1 the most frequent. The IIa subtype family is considered prevalent in calves in South America. Subtypes IIaA17G2R1 and IIaA17G4R1 had been worldwide distribution. As all C. parvum subtypes observed in calves in Chile were isolated from diarrheic animals, so, it can be possible to relate its presence with the pathogenic role in the bovine host and with a potential digestive disease risk for humans.

Background
Cryptosporidium parvum (Protozoan, Apicomplexa) is the most important eukaryotic unicellular pathogen causing diarrhea in calves worldwide [1] and is one of the two leading causes of human cryptosporidiosis [2]. Acute diarrheic calves present lethargy, anorexia, fever accompanied by dehydration, collapse and death [3]. Furthermore, infection of dairy heifers results in less milk production due to nutrition complications such as nutrient malabsorption [4]. Bovine meat production is also impacted as cryptosporidiosis in pre-weaned calves results in lower average daily gain weight [5]. Cryptosporidium oocysts excreted by infected calves can contaminate the environment, facilitating transmission of the disease by fecal-oral route not only between animals but also to humans [6]. Indeed, cattle is the most important source of zoonotic Cryptosporidium [7]. Contaminated watersheds are an important source of Cryptosporidium infection to other animals [8] as well as to humans [9], and especially in developing countries where irrigation systems include rivers with scarce infrastructure for preventing fecal contamination [10]. Molecular identi cation of C. parvum isolates throughout GP60 based approach has been used widely to study the structure of the parasite populations and its dynamics of transmission in calves [11]. The GP60 gene has nucleotide variation greater than the average in the genome of Cryptosporidium and its alleles are used to de ne groups (subtype families) among the different isolates [12]. Calves are frequently infected by the C. parvum IIa subtype family. A subtype, IIaA15G2R1 is considered highly pathogenic and is the most common infecting calves worldwide [13], meanwhile in Europe, Asia and Egypt the IId subtype family is mostly observed infecting these animals [14]. The main objective of the present work was to molecularly study the epidemiology of bovine cryptosporidiosis in Chile, by characterizing the GP60 subtypes of C. parvum infecting diarrheic dairy calves from two geographically distinct dairy zones.

Results
Fifty percent (50%) of the samples presented microscopically Cryptosporidium oocysts, 18 samples from MR and 18 samples from LRR. From these samples, the genus speci c SSU-rDNA PCR for Cryptosporidium was positive in 29 isolates and only 15 (51.7%) were GP60 positive PCR, of which 5 were from MR and 10 from LLR. Three C. parvum subtypes belonging to IIa subtype family were observed in the MR: IIaA15G2R1, IIaA17G2R1 and IIaA17G4R1. In the LRR, the subtype IIaA15G2R1 was observed in the 100% of the bovines parasite samples (Table 1).
NGS analysis of a single selected DNA sample of our study showed similarly the predominant Sanger IIaA17G4R1 GP60 subtype in 90% of the readable sequences along with others less frequent subtypes ( Table 2)

Discussion
Of the 29 SSU-rDNA PCR Cryptosporidium positive samples only 51.7% were positive to GP60. The GP60 gene has a unique copy in the Cryptosporidium genome [15] instead of SSU-rDNA gene that possess ve copies [16] making it a less sensitive in a PCR assay. Pre-weaning cattle are the most susceptible to infection especially by C. parvum [17], but it has been observed other parasites species such as C. bovis, C. ryanae and C. andersoni that could explain the lower number of positive samples by PCR in relation to the microscopy morphological tests.
The GP60 amplicons were sequenced all belonging to IIa subtype family (Table 1). Interestingly, in the LRR, the subtype IIaA15G2R1 was observed in the 100% of the samples. C. parvum subtype IIa predominates in calves in South America, in countries such as Argentina, Colombia and Brazil [18][19][20]. In Chile, IIaA15G2R1 predominates in the 86.6% of the samples which agrees with data from other countries studies. Feng et al. (2013) [21] described that the IIaA15G2R1 subtype has a high rate of transmissibility as an adaptive characteristic. IIaA17G2R1 has also been described in cattle in Europe and USA. The subtype IIaA17G4R1 has also been observed in Colombia, from diarrheic calves [20].
Although subtype diversity was observed in the samples, the predominant subtype was IIaA15G2R1 in both geographical regions of Chile, suggesting its highly infective characteristic. Most of the infections in neonatal diarrheic calves in LRR can be consequence of the biogeographic characteristics of the region, with large number of surface watercourses [22,23].
Interestingly, the NGS analysis of a single selected DNA sample of our study showed similarly the predominant IIaA17G4R1 GP60 subtype in 90% of the readable sequences along with others less frequent subtypes. This result is presented con rming by using the NGS approach that multiple subtypes of C. parvum are present naturally in an infected host as reported before [24].

Conclusions
A general conclusion is that in two different biogeographical regions of Chile, cryptosporidiosis in neonatal calves is caused by C. parvum of limited number of subtypes. The main parasite subtype is IIaA15G2R1, which is the subtype in cattle mostly reported worldwide. The presence of C. parvum in Chile is a potential risk of infection for humans, especially for dairy farm workers and veterinarians, who are in most contact with infected animals. This study contributes to a better understanding of the dynamics of cryptosporidiosis transmission in Chile also in South America and globally.

Consent for publication
Not applicable.

Competing interest
The authors declare that they have no competing interest.

Availability of data and materials
The datasets used and analyzed for this study are available from the corresponding author on reasonable request. Authors' contributions SP carried out the DNA isolation, performed PCR, bioinformatics analysis and drafted the manuscript. PM, ER and FF contributed to recollect part of the samples and revised the manuscript. LSO performed PCR, bioinformatics analysis and revised the manuscript. RM conceive the study and design, perform microscopy examination, carried out bioinformatics analysis and drafted the manuscript. All authors read and approved the nal manuscript.