Molecular analysis of Cryptosporidium species in domestic goat in central Iraq

Introduction

Cryptosporidiosis disease has been considered a global problem in humans and animals, caused by coccidian protozoan parasite Cryptosporidium species (1,2). The most prevalent species of the parasite present in ruminants are C. parvum, C. xiaoi, C. bovis, C. andersoni, C. ryanae, and C. ubiquitum (3). The common gene used for the identification and characterization of Cryptosporidium species is a heat shock protein 70 (HSP70) and glycoprotein (gp60) genes (4). The (HSP70) gene belongs to multigene families and is highly conserved across the eukaryotes and prokaryotes. Hsp70 protects cells and keeps them alive when exposed to various stress conditions (5-7). The gp60 gene is commonly used for subtyping Cryptosporidium species and is attributable to having tandem iterate of the trinucleotide coding- serine TCG, TCT, or TCA at the 50 (gp40) end of the locus (8). 

Materials and methods

One hundred fecal samples were collected from diseased and clinically healthy domestic goats from June 2020 to January 2021. The samples were collected from domestic goat farms located in four districts in Babylon city (Hilla city, Al-Hashmiyah, Mahaweel, and Al-Musayyib). The samples were collected directly from the rectum using gloves and plastic containers.

Phenotypic identification of Cryptosporidium species

Fecal samples were investigated to detect Cryptosporidium oocysts using the Ziehl Neelsen stain smears method (9). We will take 50 positive samples for molecular examination.

DNA extraction

DNA was extracted from fecal samples using AddPrep Genomic DNA Extraction Kit (addbio, Daejeon, Korea) according to the manufacturer’s instruction (9).

Nested PCR

The nested PCR method was used to detect Cryptosporidium species by targeting the (HSP70) gene, and the same reaction was used to diagnose the subtypes of C. hominins and C. parvum by targeting the (gp60) gene. Various primers were used for each process, as shown in (Table 1). 

Table 1: Oligonucleotide primer sequences used for Cryptosporidium species PCR methods.

Statistical analysis

The Chi-square test was used to compare the results. Differences were considered statistically significant at P<0.05.

Result

Detection of genotyping of Cryptosporidium spp.

The results of nested PCR for detection of Cryptosporidium spp. and the bands appear on the agarose gel (Figure1). They are sent to the sequencing to know the Cryptosporidium spp. in goats. The sequencing results for Cryptosporidium spp. showed different ratios between the species, with C. parvum being the most common. The total percentage of C. parvum in goats was 46.15% of the examined with the accession number (MZ787781, MZ787782, MZ787783, MZ787784, MZ787785, and MZ787786). The total percentage of C. hominis and C. Xiao in goats was 23.07% of the samples sent, with the accession number (MZ787787, MZ787788, MZ787789, MZ787791, MZ787792, and MZ787793) respectively. The lowest common is C. ryanae and C. bovis, which were recorded with a rate of 07.14% with the accession number (MZ787790 and MZ787794), respectively.

Detection of subtype of C. parvum and C. hominis

The results of nested PCR for C. parvum and C. hominis and the bands that appear on the agarose gel are sent to the sequencing to know the C. hominis and C. parvum ​subtype (Figure 2) in goats. C. parvum alignments with reference sequences classify all isolates into one family: IId. Extra sub-classification led to five different subtypes, the subtype IIdA21G1 being the most common, present in six cases in goats with accession numbers (MZ787819, MZ787820, MZ787821, MZ787822. MZ787823, and MZ787824) and subtype IIdA19G1 identified in four cases in goats, it has accession number (MZ787815, MZ787816, MZ787817, and MZ787818). C. hominis alignments with reference sequences classify all isolates into one family: Ib. Extra sub-classification led to 3 subtypes was detected with IbA21G2 subtype is common, found in six cases in goats with accession numbers (MZ787825, MZ787826, MZ787827, MZ787828, MZ787829, and MZ787830). Subtype IbA13G3 was identified in four cases in goats with accession numbers (MZ787831, MZ787832, MZ787833, and MZ787834), and subtype IbA19G2 was identified in two cases in goats with the accession numbers (MZ787835 and MZ787836).

Figure 1: Gel electrophoresis 1% shows some positive PCR in fecal samples of goats (size= 448 bp) using safe gel stain dye and using universal primers (first round). M is a molecular marker (100- 1100 bp) from (ADDBIO, Korea); C is a control negative.

Figure 2: Gel electrophoresis 1% using species-specific primers, using safe gel stain dye shows some positive amplicons for the subtypes of Cryptosporidium hominis (size= 800 bp). 

Phylogenetic characterization

14 Cryptosporidium sequences were evaluated to understand the relationship of Cryptosporidium species isolated in the present study. A Six C. parvum, three isolates for both C. hominis and C. xiaoi, and one isolate for both C. ryanae and C. bovis had been checked in the GenBank database in accession no. MZ787781 to MZ787794. There is closely 100% of similarity/ Sequence homology) related between C. hominis and C. parvum. The C. parvum isolate was the same as the C. parvum isolate from the Netherlands under accession no ABD60355.1, and the C. hominis isolates were the same as the C. hominis isolate from Brazil under accession no AMR08234.1 (Figure 3). 

Figure 3: Phylogenetic tree analysis of Cryptosporidium spp. created on the sequence of the Hsp70 gene of goat isolates used for local Cryptosporidium spp. (referred to as circular) compared with global Cryptosporidium spp. (referred to as triangle).

Ten isolates of the C. parvum subtype had been checked in GeneBank base data in accession no. MZ787815 - MZ787824, twelve isolates for C. hominis subtype, were checked in GeneBank base data in accession no. MZ787825 - MZ787836. C. parvum IIdA21G1 subtype isolate was the same as the C. parvum isolate from Italy under accession no. ALH22624.1 and the C. parvum IIdA19G1 subtype isolate were identical to the C. hominis isolate from China under accession no. QIC04069.1. C. hominis IbA13G3 subtype isolate was the same as C. hominis isolate from Australia under accession no. AEN71166.1 and UK under accession no. ADK92641.1, the C. hominis IbA19G2, and IbA32G2 subtype showed high relation (Figure 4)