Prevalence and molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis in dairy cattle in Ningxia, northwestern China.

Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are common intestinal pathogens that infect humans and animals. To date, research regarding these three protozoa in the Ningxia Hui Autonomous Region (Ningxia) has mostly been limited to a single pathogen, and comprehensive data on mixed infections are unavailable. This study aimed to evaluate the zoonotic potential of these three protozoa. In this study, small subunit ribosomal RNA (SSU rRNA) and 60 kDa glycoprotein (gp60) genes of Cryptosporidium; internal transcribed spacer (ITS) gene of E. bieneusi; and SSU rRNA, glutamate dehydrogenase (gdh), triosephosphate isomerase (tpi), and beta-giardin (bg) genes of G. duodenalis were examined. DNA extraction, polymerase chain reaction, and sequence analysis were performed on fecal samples collected from 320 dairy cattle at three intensive dairy farms in Ningxia in 2021 to determine the prevalence and genetic characteristics of these three protozoa. The findings revealed that 61.56% (197/320) of the samples were infected with at least one protozoan. The overall prevalence of Cryptosporidium was 19.38% (62/320), E. bieneusi was 41.56% (133/320), and G. duodenalis was 29.38% (94/320). This study identified four Cryptosporidium species (C. bovis, C. andersoni, C. ryanae, and C. parvum) and the presence of mixed infections with two or three Cryptosporidium species. C. bovis was the dominant species in this study, while the dominant C. parvum subtypes were IIdA15G1 and IIdA20G1. The genotypes of E. bieneusis were J, BEB4, and I alongside the novel genotypes NX1-NX8, all belonging to group 2, with genotype J being dominant. G. duodenalis assemblages were identified as assemblages E, A, and B, and a mixed infection involving assemblages A + E was identified, with assemblage E being the dominant one. Concurrently, 11 isolates formed 10 different assemblage E multilocus genotypes (MLGs) and 1 assemblage A MLG and assemblage E MLGs formed 5 subgroups. To the best of our knowledge, this is the first report on mixed infection with two or three Cryptosporidium species in cattle in Ningxia and on the presence of the C. parvum subtype IIdA20G1 in this part of China. This study also discovered nine genotypes of E. bieneusis and novel features of G. duodenalis assemblages in Ningxia. This study indicates that dairy cattle in this region may play a significant role in the zoonotic transmission of Cryptosporidium spp., E. bieneusi, and G. duodenalis.

Cloning and characterization of OsORC2, a new member of rice origin recognition complex.

In eukaryotic cells, the origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. The isolation, characterization and tissue-specific expression of a putative ORC subunit 2 (OsORC2) in Oryza sativa is described here. A novel cDNA fragment encoding rice ORC2 was isolated by screening the subtractive library, which had a higher expression level in inflorescence meristem than in shoot apical meristem. The full-length cDNA of rice ORC2 was obtained by the method of rapid amplification of cDNA ends, which contained an 1140 bp open reading frame encoding a 379 amino acid polypeptide. Sequence alignment shows that there is a high homology between the deduced amino sequence of OsORC2 and maize ORC2 (85%). The tissue-specific expression pattern of OsORC2 reveals that it is abundant in roots, seedling and inflorescence meristem, while its expression level is much lower in mature leaves and shoot.

[Isolation and analysis of a novel MYC gene from rice].

A MYC (Myelocytomatosis) transcription factor gene, OsMYC, was cloned in rice (Oryza sativa L.). The putative protein of the OsMYC gene has a typical DNA binding domain: basic/helix-loop-helix (bHLH) motif at the C-terminus. Its nucleocide sequnce has 78%, 48%, 46% identity with that of the AtMYC2, MYC7E and PG1 genes, respectively. The identity of deduced amino acid between OsMYC and AtMYC2, MYG7E and PG1 is 95%, 84%, and 77% in the bHLH domain, and that is 81%, 54% and 52% in the N-terminus conserved region. The identity is 100% in the nuclear localization signal between them. Phylogenetic comparisons revealed that the OsMYC protein was related to the Arabidopsis AtMYC2, Zea MYC7E and Phaseolus vulgaris PG1. OsMYC gene was expressed at high level in stem, but low level in leaf and root and can be induced by ABA and Fe3+, which is similar to the expression pattern of MYC7E and RAP1 genes. Thus, this gene is a new member of the rice MYC family.

Isolation, characterization and expression analysis of a leaf-specific phosphoenolpyruvate carboxylase gene in Oryza sativa.

Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.

Cloning, characterization and prokaryotic expression of cytosolic malate dehydrogenase from Oryza sativa.

cDNA sequences of malate dehydrogenase (MDH) were cloned from various species, and MDH was identified to play an important role in cell energy metabolism. Here, we present the isolation and characterization of its homologue (OscMDH) in Oryza sativa. Comparison of the results to the genome details indicated that OscMDH consisted of seven exons. Sequence alignment showed that the deduced amino acid sequence of OscMDH shared a significant similarity with cMDH protein in Zea mays, as well as with other cMDH gene products. The different expression patterns of OscMDH in different tissues revealed that OscMDH mRNA was highly transcribed in either young panicle or immature seed, while its abundance was much low in green leaf and root. A nearly 56-kDa fusion protein generated by adding a Trx-His-tag at the N-terminal of OscMDH was induced by IPTG in Escherichia coli BL21 and an obvious MDH activity was detected in the protein by native PAGE analysis. All these results suggest that OscMDH encodes a cytosolic MDH in rice.

[The cloning and expression of a novel rPDCD5 gene from rice].

An EST related to the gene PCD was isolated from SSH (suppression subtractive hybridization) library of callus tissues of rice (Oryza sativa L.). Primers were designed to obtain its complete cDNA encoding putative apoptosis-related protein from Shanyou 63 (Oryza sativa L.). Sequencing indicated that the gene contained a 387 bp open reading frame, which encodes a protein containing 128 aa. Sequence alignment showed that the deduced protein is highly homologous to the known PDCD5. Real time quantitative PCR was performed to reveal that rPDCD5 was up-regulated in abiotic stress (low temperature and NaCl treatment).

Cloning and characterization of a novel Hsp100/Clp gene (osClpD) from Oryza sativa.

A novel osClpD gene, encoding a highly conservative ClpD subfamily member, was first isolated and characterized from Oryza sativa. The full-length cDNA of osClpD gene was 3140 bp and contained a 2884 bp open reading frame encoding a 938 amino acid protein. The phylogenetic tree and blast search showed that OSClpD belonged to the ClpD subfamily of the Hsp100/Clp family, and contained all protein motifs characteristic for the ClpD subfamily of Hsp100/Clp proteins. The real-time quantitative PCR analysis proved that it was inducible by water deficit and temperature stress in vegetative tissues.

cDNA cloning and expression analysis of the rice (Oryza sativa L.) RNase L inhibitor.

A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.

Cloning and prokaryotic expression of a cDNA encoding a putative mitochondrial malate dehydrogenase in Oryza sativa.

Malate dehydrogenase (MDH) has been characterized as a key player in oxaloacetate (OAA) biosynthesis mechanism in citrate acid cycle that generates reducing powers for further assimilation in the whole cell. Here we present the cloning, characterization and prokaryotic expression of a putative Mdh (OsmMDH) in Oryza sativa. Sequence alignment shows that there is a high homology between the deduced amino acid sequence of OsmMDH and MDH portein in Eucalyptus gunnii (80%), as well as between the deduced amino acid sequence of OsmMDH and other MDHs. Moreover, pI and the mitochondrial location of OsmMDH are predicted. The tissue-specific expression pattern of OsmMDH reveals that it is abundant in young panicle and immature seed, while its expression level is mush lower in leaf and root. Its expression in E. coli BL21 as a fusion gene is studied further.